Antisense oligonucleotide compositions

ABSTRACT

The present disclosure provides compositions comprising an antisense oligonucleotide and one or more excipients that modulates viscosity, turbidity or both viscosity and turbidity. In certain embodiments, compositions comprising an antisense oligonucleotide and one or more excipients having low viscosity are provided. In certain embodiments, compositions comprising an antisense oligonucleotide and one or more excipients having low turbidity are provided. In certain embodiments, pharmaceutical compositions comprising an antisense oligonucleotide and one or more excipients having low viscosity and turbidity are provided.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0105USC2SEQ.txt, created Nov. 28, 2018, which is 8 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Antisense compounds have been used to modulate target nucleic acids.Antisense compounds comprising a variety of chemical modifications andmotifs have been reported. In certain instances, such compounds areuseful as research tools, diagnostic reagents, and as therapeuticagents. Certain DNA-like oligomeric compounds have been shown to reduceprotein expression. Certain RNA-like compounds are known to inhibitprotein expression in cells. Such RNA-like compounds function, at leastin part, through the RNA-inducing silencing complex (RISC). RNA-likecompounds may be single-stranded or double-stranded. Antisense compoundshave also been shown to alter processing of pre-mRNA and to modulatenon-coding RNA molecules. In certain instances antisense compounds havebeen shown to modulate protein expression by binding to a targetmessenger RNA (mRNA) encoding the protein. In certain instances, suchbinding of an antisense compound to its target mRNA results in cleavageof the mRNA. Antisense compounds that modulate processing of a pre-mRNAhave also been reported. Such antisense compounds alter splicing,interfere with polyadenlyation or prevent formation of the 5′-cap of apre-mRNA. Compositions and methods that increase productive uptake ofantisense compounds in cells are desired. Compositions and methods thatfacilitate the manufacture, storage, administration, and delivery ofantisense compounds are also desired.

SUMMARY OF THE INVENTION

The present disclosure provides compositions and methods that facilitatethe manufacture, storage, administration, and delivery of antisenseoligonucleotide solutions. For example, in certain embodiments, thepresent disclosure provides a number of excipients that reduce theviscosity of an antisense oligonucleotide solution. In certainembodiments, the present disclosure provides a number of excipients thatreduce the turbidity of an antisense oligonucleotide solution. Incertain embodiments, the present disclosure provides a number ofexcipients that reduce both the turbidity and the viscosity of anantisense oligonucleotide solution. In certain embodiments, the presentdisclosure provides a number of excipients that modulate viscosity,turbidity or both viscosity and turbidity of antisense oligonucleotidesolutions wherein the antisense oligonucleotide is present in thesolution at a high concentration. In certain embodiments, the presentdisclosure provides a number of excipients that reduce both theturbidity and the viscosity of an antisense oligonucleotide solution andwherein the excipient also modulates the osmolality of the antisenseoligonucleotide solution. In certain embodiments, the present disclosureprovides a number of excipients that modulate viscosity, turbidity, orosmolality or viscosity and turbidity and osmolality of an antisenseoligonucleotide solution and wherein the antisense oligonucleotide ispresent in the solution at a high concentration.

The present disclosure provides the following non-limiting numberedembodiments:

Embodiment 1. An aqueous solution comprising:

-   -   an antisense oligonucleotide; and    -   at least one excipient that modulates viscosity, turbidity or        both viscosity and turbidity;    -   wherein the dynamic viscosity of the solution is less than 100        cP at at 25° C.

Embodiment 2. The solution of embodiment 1, having a dynamic viscosityof less than 90 cP at 25° C.

Embodiment 3. The solution of embodiment 1, having a dynamic viscosityof less than 80 cP at 25° C.

Embodiment 4. The solution of embodiment 1, having a dynamic viscosityof less than 70 cP at 25° C.

Embodiment 5. The solution of embodiment 1, having a dynamic viscosityof less than 60 cP at 25° C.

Embodiment 6. The solution of embodiment 1, having a dynamic viscosityof less than 50 cP at 25° C.

Embodiment 7. The solution of embodiment 1, having a dynamic viscosityof less than 40 cP at 25° C.

Embodiment 8. The solution of embodiment 1, having a dynamic viscosityof less than 30 cP at 25° C.

Embodiment 9. The solution of embodiment 1, having a dynamic viscosityof less than 20 cP at 25° C.

Embodiment 10. The solution of embodiment 1, having a dynamic viscosityof less than 10 cP at 25° C.

Embodiment 11. The solution of any of embodiments 1-10, having aturbidity of in the range of 0 to 4000 NTU.

Embodiment 12. The solution of any of embodiments 1-10, having aturbidity of in the range of 0 to 3000 NTU.

Embodiment 13. The solution of any of embodiments 1-10, having aturbidity of in the range of 0 to 2000 NTU.

Embodiment 14. The solution of any of embodiments 1-10, having aturbidity of in the range of 0 to 1000 NTU.

Embodiment 15. The solution of any of embodiments 1-10, having aturbidity of in the range of 0 to 100 NTU.

Embodiment 16. The solution of any of embodiments 1-10, having aturbidity of in the range of 0 to 90 NTU.

Embodiment 17. The solution of any of embodiments 1-10, having aturbidity of the solution is in the range of 0 to 80 NTU.

Embodiment 18. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 70 NTU.

Embodiment 19. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 60 NTU.

Embodiment 20. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 50 NTU.

Embodiment 21. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 40 NTU.

Embodiment 22. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 30 NTU.

Embodiment 23. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 25 NTU.

Embodiment 24. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 20 NTU.

Embodiment 25. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 15 NTU.

Embodiment 26. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 10 NTU.

Embodiment 27. The solution of any of embodiments 1-10, having aturbidity in the range of 0 to 5 NTU.

Embodiment 28. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 1 mg/ml to500 mg/ml.

Embodiment 29. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 1 to 400mg/mL.

Embodiment 30. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 1 to 300mg/mL.

Embodiment 31. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 1 to 280mg/mL.

Embodiment 32. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 1 to 260mg/mL.

Embodiment 33. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 1 to 240mg/mL.

Embodiment 34. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 1 to 220mg/mL.

Embodiment 35. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 1 to 200mg/mL.

Embodiment 36. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 100 to 220mg/mL.

Embodiment 37. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 120 to 220mg/mL.

Embodiment 38. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 140 to 220mg/mL.

Embodiment 39. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 160 to 220mg/mL.

Embodiment 40. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 180 to 220mg/mL.

Embodiment 41. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 130 to 170mg/mL.

Embodiment 42. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 135 to 165mg/mL.

Embodiment 43. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 140 to 160mg/mL.

Embodiment 44. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 145 to 155mg/mL.

Embodiment 45. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 150 mg/mL.

Embodiment 46. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 50 mg/mL.

Embodiment 47. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 40 mg/mL.

Embodiment 48. The solution of any of embodiments 1 to 27, wherein theantisense oligonucleotide is present at a concentration of 60 mg/mL.

Embodiment 49. The solution of any of embodiments 1 to 48, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity comprises an aromaticcompound.

Embodiment 50. The solution of any of embodiments 1 to 48, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is an aromatic compound.

Embodiment 51. The solution of any of embodiments 1 to 50, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is a heterocyclic compound.

Embodiment 52. The solution of any of embodiments 1 to 51, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is a heterocyclic aromaticcompound.

Embodiment 53. The solution of any of embodiments 1 to 50, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity comprises a heterocycliccompound.

Embodiment 54. The solution of embodiment 51 or 52, wherein theheteroatom of the heterocyclic compound is nitrogen.

Embodiment 55. The solution of embodiment 51 or 52, wherein theheteroatom of the heterocyclic compound is oxygen.

Embodiment 56. The solution of embodiment 51 or 52, wherein theheteroatom of the heterocyclic compound is not oxygen.

Embodiment 57. The solution of embodiment 51 or 52, wherein theheteroatom of the heterocyclic compound is sulfur.

Embodiment 58. The solution of any of embodiments 51 to 57, whereinheterocyclic compound has more than one heteroatom.

Embodiment 59. The solution of any of embodiments 51 to 57, whereinheterocyclic compound has two or more heteroatoms.

Embodiment 60. The solution of any of embodiments 51 to 59, whereinheterocyclic compound comprises a monocyclic ring having two or moreheteroatoms.

Embodiment 61. The solution of any of embodiments 51 to 59, whereinheterocyclic compound is a heterocyclic aromatic compound that comprisesa monocyclic ring having two or more heteroatoms.

Embodiment 62. The solution of any of embodiments 60 to 61 where theheteroatoms are different.

Embodiment 63. The solution of any of embodiments 60 to 61 where theheteroatoms are the same.

Embodiment 64. The solution of embodiment 60 or 63, wherein theheteroatom of the heterocyclic compound is nitrogen.

Embodiment 65. The solution of embodiment 60 or 63, wherein theheteroatom of the heterocyclic compound is oxygen.

Embodiment 66. The solution of embodiment 60 or 63, wherein theheteroatom of the heterocyclic compound is not oxygen.

Embodiment 67. The solution of embodiment 60 or 63, wherein theheteroatom of the heterocyclic compound is sulfur.

Embodiment 68. The solution of any of embodiments 1 to 52, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is a pyridine.

Embodiment 69. The solution of any of embodiments 1 to 52, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is a diazine.

Embodiment 70. The solution of embodiment 69, wherein the diazine is1,2-diazine.

Embodiment 71. The solution of embodiment 69, wherein the diazine is1,3-diazine.

Embodiment 72. The solution of embodiment 69, wherein the diazine is1,4-diazine.

Embodiment 73. The solution of any of embodiments 1 to 52, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is an imidazole.

Embodiment 74. The solution of any of embodiments 1 to 52, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is a tetrazine.

Embodiment 75. The solution of any of embodiments 1 to 52, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is a hexazine.

Embodiment 76. The solution of embodiment 53, wherein the heterocycliccompound comprises a heterocyclic amine.

Embodiment 77. The solution of any of embodiments 1 to 69, wherein theexcipient is a hydrogen bond doner.

Embodiment 78. The solution of any of embodiments 1 to 69, wherein theexcipient is a hydrogen bond acceptor.

Embodiment 79. The solution of any of embodiments 1 to 78, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidityis selected from the groupconsisting of Adenine, Benzyl Alcohol, m-Cresol, Cytidine, CytidineMonophosphate, Cytosine, Dextran, Guanine Monophosphate, D-Mannitol,Methylparaben, Nicotinamide, Phenol, 2-Phenoxyethanol, L-Phenylalanine,Pyridoxine, Sodium Chloride, Thymine, tryptophan, L-Tryptophan,L-Tyrosine, Ascorbic Acid, Benzamide, o-Benzenediol, Benzenehexol,L-Histidine, Hydroxypyridine, Indole, D-Mannitol+Phenol mixture,D-Mannitol+Pyridine mixture, 2H-Pyran, Pyrazinamide, Pyridine,Pyrimidine, 2-Pyrone, Riboflavin, Thiamine, Tryptamine, ethanol,(2-Hydroxypropyl)-β-cyclodextrin, Niacin, Polyethylene Glycol 600,Polyethylene Glycol 4600, Propylene Glycol, Pyridoxine, Sucrose,Thymidine, Tween 80, Uridine, Thymine, caffeine, acridine orange,ethidium bromide, propidium iodide, cyanine dyes such as PicoGreen®,thiamine hydrochloride, ethylene diamine tetraacetic acid,1,2-dihydroxybenzene, 1,2-dihydroxybenzene (catechol), D-Mannitol+phenolmixture, D-Mannitol+niacinamide mixture, calcium folinate,L-phenylalanine+pyridoxine mixture, pyridoxine+benzyl alcohol mixture,pyridoxine HCl+benzyl alcohol mixture, niacin sodium salt, dextran 1500,and pyridoxine HCl+phenylalanine mixture.

Embodiment 80. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Adenine.

Embodiment 81. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Benzyl Alcohol.

Embodiment 82. The solution of embodiment 79, wherein at least one ofthe one or more excipient is m-Cresol.

Embodiment 83. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Cytidine.

Embodiment 84. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Cytidine Monophosphate.

Embodiment 85. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Cytosine.

Embodiment 86. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Dextran.

Embodiment 87. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Guanine Monophosphate.

Embodiment 88. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is D-Mannitol.

Embodiment 89. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Methylparaben.

Embodiment 90. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Nicotinamide.

Embodiment 91. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Phenol.

Embodiment 92. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is 2-Phenoxyethanol.

Embodiment 93. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is L-Phenylalanine.

Embodiment 94. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Pyridoxine.

Embodiment 95. The solution of embodiment 79, wherein at least one of atleast one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Sodium Chloride.

Embodiment 96. The solution of embodiment 79, wherein at least one of atleast one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Thymine.

Embodiment 97. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is L-Tryptophan.

Embodiment 98. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is tryptophan.

Embodiment 99. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is L-Tyrosine.

Embodiment 100. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Ascorbic Acid.

Embodiment 101. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Benzamide.

Embodiment 102. The solution of embodiment 79, wherein at least one ofthe one or more excipient is o-Benzenediol.

Embodiment 103. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Benzenehexol.

Embodiment 104. The solution of embodiment 79, wherein at least one ofthe one or more excipient is L-Histidine.

Embodiment 105. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is a hydroxypyridine.

Embodiment 106. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is an indole.

Embodiment 107. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is D-Mannitol.

Embodiment 108. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is a D-Mannitol and phenol mixture.

Embodiment 109. The solution of embodiment 79, wherein at least one ofat least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is a D-Mannitol and pyridine mixture.

Embodiment 110. The solution of embodiment 79, wherein at least one ofat least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is 2H-Pyran.

Embodiment 111. The solution of embodiment 79, wherein at least one ofat least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is a pyrazinamide.

Embodiment 112. The solution of embodiment 79, wherein at least one ofat least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is a pyridine.

Embodiment 113. The solution of embodiment 79, wherein at least one ofat least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is a pyrimidine.

Embodiment 114. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is 2-Pyrone.

Embodiment 115. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is riboflavin.

Embodiment 116. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is thiamine.

Embodiment 117. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is tryptamine.

Embodiment 118. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is ethanol.

Embodiment 119. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is (2-Hydroxypropyl)-β-cyclodextrin.

Embodiment 120. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Niacin.

Embodiment 121. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Polyethylene Glycol 790.

Embodiment 122. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Polyethylene Glycol 4790.

Embodiment 123. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Propylene Glycol.

Embodiment 124. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Pyridoxine.

Embodiment 125. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Sucrose.

Embodiment 126. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Thymidine.

Embodiment 127. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Tween 80.

Embodiment 128. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Uridine.

Embodiment 129. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is Thymine.

Embodiment 130. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is caffeine.

Embodiment 131. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is acridine orange.

Embodiment 132. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is ethidium bromide.

Embodiment 133. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is propidium iodide.

Embodiment 134. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is a cyanine dye.

Embodiment 135. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is PicoGreen®.

Embodiment 136. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is 1,2-dihydroxybenzene (catechol).

Embodiment 137. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is D-Mannitol+phenol mixture.

Embodiment 138. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is D-Mannitol+niacinamide mixture.

Embodiment 139. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is calcium folinate.

Embodiment 140. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is L-phenylalanine+pyridoxine mixture.

Embodiment 141. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is pyridoxine+benzyl alcohol mixture.

Embodiment 142. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is pyridoxine HCl+benzyl alcohol mixture.

Embodiment 143. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is pyridoxine HCl+phenylalanine mixture.

Embodiment 144. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is D-Mannitol (0.5% w/v)+phenol (0.5% w/v)mixture.

Embodiment 145. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is D-Mannitol (0.5% w/v)+niacinamide (0.5% w/v)mixture.

Embodiment 146. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is D-Mannitol (0.5% w/v)+pyridine (0.5% w/v)mixture.

Embodiment 147. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is L-phenylalanine (1.0% w/v)+pyroxidine (0.5%w/v) mixture.

Embodiment 148. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is pyroxidine (0.5% w/v)+benzyl alcohol (0.5%w/v) mixture.

Embodiment 149. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is pyroxidine HCl (0.5% w/v)+benzyl alcohol(0.5% w/v) mixture.

Embodiment 150. The solution of embodiment 79, wherein at least one ofthe at least one excipient that modulates viscosity, turbidity or bothviscosity and turbidity is pyroxidine HCl (0.5% w/v)+phenylalanine (1.0%w/v) mixture.

Embodiment 151. The solution of any of embodiments 1 to 150, wherein thesolution does not comprise cyclodextrin.

Embodiment 152. The solution of any of embodiments 1 to 150, wherein thesolution does not comprise mannitol.

Embodiment 153. The solution of any of embodiments 1 to 152, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is an antimicrobial.

Embodiment 154. The solution of any of embodiments 1 to 153, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity is not an antimicrobial.

Embodiment 155. The solution of any of embodiments 1 to 152 or 154,wherein the solution does not comprise any excipient that is anantimicrobial.

Embodiment 156. The solution of any of embodiments 1 to 155, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity increases the osmolarity ofthe solution.

Embodiment 157. The solution of any of embodiments 1 to 156, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity increases the pH of thesolution.

Embodiment 158. The solution of any of embodiments 1 to 156, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity decreases the pH of thesolution.

Embodiment 159. The solution of any of embodiments 1 to 158, wherein atleast one of the at least one excipient that modulates viscosity,turbidity or both viscosity and turbidity buffers the pH of thesolution.

Embodiment 160. The solution of any of embodiments 1 to 159, wherein thesolution comprises only one excipient that modulates viscosity,turbidity or both viscosity and turbidity.

Embodiment 161. The solution of any of embodiments 1 to 160, wherein thesolution comprises only one excipient.

Embodiment 162. The solution of any of embodiments 1 to 160, wherein thesolution comprises at least two excipients.

Embodiment 163. The solution of any of embodiments 1 to 160, wherein thesolution comprises at least three excipients.

Embodiment 164. The solution of any of embodiments 1 to 160, wherein thesolution comprises at least four excipients.

Embodiment 165. The solution of any of embodiments 1 to 160, wherein thesolution comprises at least two excipients that modulate viscosity,turbidity or both.

Embodiment 166. The solution of any of embodiments 1 to 160, wherein thesolution comprises at least three excipients that modulate viscosity,turbidity or both.

Embodiment 167. The solution of any of embodiments 1 to 160, wherein thesolution comprises at least four excipients that modulate viscosity,turbidity or both.

Embodiment 168. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient in the solution is in the range of 0.1 to 7parts by weight.

Embodiment 169. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient in the solution is in the range of 0.3 to 7parts by weight.

Embodiment 170. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient in the solution is in the range of 0.5 to 5parts by weight.

Embodiment 171. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient in the solution is in the range of 1 to 5parts by weight.

Embodiment 172. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient in the solution is in the range of 2 to 5parts by weight.

Embodiment 173. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient in the solution is in the range of 3 to 5parts by weight.

Embodiment 174. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient in the solution is in the range of 4 to 5parts by weight.

Embodiment 175. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient in the solution is in the range of 4.5 to 5.5parts by weight.

Embodiment 176. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient that modulates viscosity, turbidity or both inthe solution is in the range of 0.1 to 7 parts by weight.

Embodiment 177. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient that modulates viscosity, turbidity or both inthe solution is in the range of 0.3 to 7 parts by weight.

Embodiment 178. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient that modulates viscosity, turbidity or both inthe solution is in the range of 0.5 to 5 parts by weight.

Embodiment 179. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient that modulates viscosity, turbidity or both inthe solution is in the range of 1 to 5 parts by weight.

Embodiment 180. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient that modulates viscosity, turbidity or both inthe solution is in the range of 2 to 5 parts by weight.

Embodiment 181. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient that modulates viscosity, turbidity or both inthe solution is in the range of 3 to 5 parts by weight.

Embodiment 182. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient that modulates viscosity, turbidity or both inthe solution is in the range of 4 to 5 parts by weight.

Embodiment 183. The solution of any of embodiments 1 to 167, wherein thetotal amount of excipient that modulates viscosity, turbidity or both inthe solution is in the range of 4.5 to 5.5 parts by weight.

Embodiment 184. The solution of any of embodiments 1 to 183, wherein theantisense oligonucleotide comprises at least one modified nucleoside.

Embodiment 185. The solution of embodiment 184, wherein at least onemodified nucleoside comprises a modified sugar moiety.

Embodiment 186. The solution of embodiment 185, wherein at least onemodified sugar moiety is a 2′-substituted sugar moiety.

Embodiment 187. The solution of embodiment 186, wherein the2′-substitutent of at least one 2′-substituted sugar moiety is selectedfrom among: 2′-OMe, 2′-F, and 2′-MOE.

Embodiment 188. The solution of embodiment 187, wherein the2′-substituent of at least one 2′-substituted sugar moiety is a 2′-MOE.

Embodiment 189. The solution of any of embodiments 1 to 188, wherein atleast one modified sugar moiety is a bicyclic sugar moiety.

Embodiment 190. The solution of embodiment 189, wherein at least onebicyclic sugar moiety is LNA or cEt.

Embodiment 191. The solution of any of embodiments 1 to 191, wherein atleast one sugar moiety is a sugar surrogate.

Embodiment 192. The solution of embodiment 191, wherein at least onesugar surrogate is a morpholino.

Embodiment 193. The solution of embodiment 191, wherein at least onesugar surrogate is a modified morpholino.

Embodiment 194. The solution of any of embodiments 1 to 193, wherein theantisense oligonucleotide comprises at least 5 modified nucleosides,each independently comprising a modified sugar moiety.

Embodiment 195. The solution of any of embodiments 1 to 193, wherein theantisense oligonucleotide comprises at least 10 modified nucleosides,each independently comprising a modified sugar moiety.

Embodiment 196. The solution of any of embodiments 1 to 193, wherein theantisense oligonucleotide comprises at least 15 modified nucleosides,each independently comprising a modified sugar moiety.

Embodiment 197. The solution of any of embodiments 1 to 196, whereineach nucleoside of the antisense oligonucleotide is a modifiednucleoside, each independently comprising a modified sugar moiety

Embodiment 198. The solution of any of any of embodiments 1 to 197,wherein the antisense oligonucleotide comprises at least two modifiednucleosides comprising modified sugar moieties that are the same as oneanother.

Embodiment 199. The solution of any of embodiments 1 to 198, wherein theantisense oligonucleotide comprises at least two modified nucleosidescomprising modified sugar moieties that are different from one another.

Embodiment 200. The solution of any of embodiments 1 to 199, wherein theantisense oligonucleotide comprises a modified region of at least 5contiguous modified nucleosides.

Embodiment 201. The solution of any of embodiments 1 to 200, wherein theantisense oligonucleotide comprises a modified region of at least 10contiguous modified nucleosides.

Embodiment 202. The solution of any of embodiments 1 to 201, wherein theantisense oligonucleotide comprises a modified region of at least 15contiguous modified nucleosides.

Embodiment 203. The solution of any of embodiments 1 to 202, wherein theantisense oligonucleotide comprises a modified region of at least 20contiguous modified nucleosides.

Embodiment 204. The solution of any of embodiments 197 to 203, whereineach modified nucleoside of the modified region has a modified sugarmoiety independently selected from among: 2′-F, 2′-OMe, 2′-MOE, cEt,LNA, morpholino, and modified morpholino.

Embodiment 205. The solution of any of embodiments 197 to 203, whereinthe modified nucleosides of the modified region each comprise the samemodification as one another.

Embodiment 206. The solution of embodiment 205, wherein the modifiednucleosides of the modified region each comprise the same 2′-substitutedsugar moiety.

Embodiment 207. The solution of embodiment 206, wherein the2′-substituted sugar moiety of the modified nucleosides of the region ofmodified nucleosides is selected from 2′-F, 2′-OMe, and 2′-MOE.

Embodiment 208. The solution of embodiment 207, wherein the2′-substituted sugar moiety of the modified nucleosides of the region ofmodified nucleosides is 2′-MOE.

Embodiment 209. The solution of embodiment 205, wherein the modifiednucleosides of the region of modified nucleosides each comprise the samebicyclic sugar moiety.

Embodiment 210. The solution of embodiment 209, wherein the bicyclicsugar moiety of the modified nucleosides of the region of modifiednucleosides is selected from LNA and cEt.

Embodiment 211. The solution of embodiment 205, wherein the modifiednucleosides of the region of modified nucleosides each comprises a sugarsurrogate.

Embodiment 212. The solution of embodiment 211, wherein the sugarsurrogate of the modified nucleosides of the region of modifiednucleosides is a morpholino.

Embodiment 213. The solution of embodiment 211, wherein the sugarsurrogate of the modified nucleosides of the region of modifiednucleosides is a modified morpholino.

Embodiment 214. The solution of any of embodiments 1 to 213, wherein themodified nucleotide comprises no more than 4 contiguous naturallyoccurring nucleosides.

Embodiment 215. The solution of any of embodiments 1 to 214, whereineach nucleoside of the antisense oligonucleotide is a modifiednucleoside.

Embodiment 216. The solution of embodiment 215 wherein each modifiednucleoside comprises a modified sugar moiety.

Embodiment 217. The solution of embodiment 216, wherein the modifiednucleosides of the antisense oligonucleotide comprise the samemodification as one another.

Embodiment 218. The solution of embodiment 217, wherein the modifiednucleosides of the antisense oligonucleotide each comprise the same2′-substituted sugar moiety.

Embodiment 219. The solution of embodiment 218, wherein the2′-substituted sugar moiety of the antisense oligonucleotide is selectedfrom 2′-F, 2′-OMe, and 2′-MOE.

Embodiment 220. The solution of embodiment 219, wherein the2′-substituted sugar moiety of the antisense oligonucleotide is 2′-MOE.

Embodiment 221. The solution of embodiment 217, wherein the modifiednucleosides of the antisense oligonucleotide each comprise the samebicyclic sugar moiety.

Embodiment 222. The solution of embodiment 221, wherein the bicyclicsugar moiety of the antisense oligonucleotide is selected from LNA andcEt.

Embodiment 223. The solution of embodiment 217, wherein the modifiednucleosides of the antisense oligonucleotide each comprises a sugarsurrogate.

Embodiment 224. The solution of embodiment 223, wherein the sugarsurrogate of the antisense oligonucleotide is a morpholino.

Embodiment 225. The solution of embodiment 223, wherein the sugarsurrogate of the antisense oligonucleotide is a modified morpholino.

Embodiment 226. The solution of any of embodiments 1 to 225, wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.

Embodiment 227. The solution of embodiment 226, wherein eachinternucleoside linkage is a modified internucleoside linkage.

Embodiment 228. The solution of embodiment 226 or 227, comprising atleast one phosphorothioate internucleoside linkage.

Embodiment 229. The solution of embodiment 227, wherein eachinternucleoside linkage is a modified internucleoside linkage andwherein each internucleoside linkage comprises the same modification.

Embodiment 230. The solution of embodiment 229, wherein eachinternucleoside linkage is a phosphorothioate internucleoside linkage.

Embodiment 231. The solution of any of embodiments 1 to 230, comprisingat least one conjugate.

Embodiment 232. The solution of any of embodiments 1 to 231, wherein theantisense oligonucleotide consists of a single-strand antisenseoligonucleotide.

Embodiment 233. The solution of any of embodiments 1 to 232, wherein theantisense oligonucleotide consists of a double-strand antisenseoligonucleotide.

Embodiment 234. The solution of any of embodiments 1 to 233, wherein theantisense oligonucleotide is selected from the group consisting of ISIS426115, ISIS 104838, ISIS 416858, ISIS 420915, ISIS 494372, ISIS 487660,ISIS 404173, ISIS 449884, ISIS 501861, ISIS 540175, ISIS 396443, ISIS463588, ISIS 301012, ISIS 329993, ISIS 304801, ISIS 426115, ISIS 481464,ISIS 183750, and ISIS 333611.

Embodiment 235. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a dynamic viscosity of more than 60 cP at25° C. when mixed in water or saline solution in the absence of anexcipient that modulates viscosity, turbidity or both viscosity andturbidity.

Embodiment 236. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a dynamic viscosity of more than 55 cP at25° C. when mixed in water or saline solution and in the absence of anexcipient that modulates viscosity, turbidity or both viscosity andturbidity.

Embodiment 237. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a dynamic viscosity of more than 50 cP at25° C. when mixed in water or saline solution and in the absence of anexcipient that modulates viscosity, turbidity or both viscosity andturbidity.

Embodiment 238. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a dynamic viscosity of more than 45 cP at25° C. when mixed in water or saline solution and in the absence of anexcipient that modulates viscosity, turbidity or both viscosity andturbidity.

Embodiment 239. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a dynamic viscosity of more than 40 cP at25° C. when mixed in water or saline solution and in the absence of anexcipient that modulates viscosity, turbidity or both viscosity andturbidity.

Embodiment 240. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a turbidity of more than 100 NTU whenmixed in water or saline solution and in the absence of an excipientthat modulates viscosity, turbidity or both viscosity and turbidity.

Embodiment 241. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a turbidity of more than 70 NTU when mixedin water or saline solution and in the absence of an excipient thatmodulates viscosity, turbidity or both viscosity and turbidity.

Embodiment 242. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a turbidity of more than 50 NTU when mixedin water or saline solution and in the absence of an excipient thatmodulates viscosity, turbidity or both viscosity and turbidity.

Embodiment 243. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a turbidity of more than 25 NTU when mixedin water or saline solution and in the absence of an excipient thatmodulates viscosity, turbidity or both viscosity and turbidity.

Embodiment 244. The solution of any of embodiments 1 to 234, wherein theantisense oligonucleotide has a turbidity of more than 20 NTU when mixedin water or saline solution and in the absence of an excipient thatmodulates viscosity, turbidity or both viscosity and turbidity.

Embodiment 245. A method of reducing the turbidity of an antisenseoligonucleotide solution, said method comprising adding an effectiveamount of an excipient that modulates viscosity, turbidity or bothviscosity and turbidity to the antisense oligonucleotide solution.

Embodiment 246. The method of embodiment 245, wherein the excipient thatmodulates viscosity, turbidity or both viscosity and turbidity is anexcipient according to any of embodiments 49-133.

Embodiment 247. The method of embodiment 245 or 246, wherein theantisense compound is an antisense compound according to any ofembodiments 184-234.

Embodiment 248. The method of any of embodiments 245-247, wherein theturbidity is reduced by 0-1000 NTU.

Embodiment 249. The method of any of embodiments 245-247, wherein theturbidity is reduced by 0-500 NTU.

Embodiment 250. The method of any of embodiments 245-247, wherein theturbidity is reduced by 0-100 NTU.

Embodiment 251. The method of any of embodiments 245-247, wherein theturbidity is reduced by 0-50 NTU.

Embodiment 252. The method of any of embodiments 245-247, wherein theturbidity is reduced by 0-30 NTU.

Embodiment 253. The method of any of embodiments 245-247, wherein theturbidity is reduced by 0-20 NTU.

Embodiment 254. The method of any of embodiments 245-247, wherein theturbidity is reduced by 0-10 NTU.

Embodiment 255. A method of reducing the viscosity of an antisenseoligonucleotide solution, said method comprising adding an effectiveamount of an excipient that modulates viscosity, turbidity or bothviscosity and turbidity to the antisense oligonucleotide solution.

Embodiment 256. The method of embodiment 255, wherein the excipient thatmodulates viscosity, turbidity or both viscosity and turbidity is anexcipient according to any of embodiments 49-155.

Embodiment 257. The method of embodiment 255 or 256, wherein theantisense compound is an antisense compound according to any ofembodiments 184-234.

Embodiment 258. The method of any of embodiments 255-257, wherein theviscosity is reduced by 1-100 cP at 25° C.

Embodiment 259. The method of any of embodiments 255-257, wherein theviscosity is reduced by 1-50 cP at 25° C.

Embodiment 260. The method of any of embodiments 255-257, wherein theviscosity is reduced by 1-40 cP at 25° C.

Embodiment 261. The method of any of embodiments 255-257, wherein theviscosity is reduced by 1-30 cP at 25° C.

Embodiment 262. The method of any of embodiments 255-257, wherein theviscosity is reduced by 1-20 cP at 25° C.

Embodiment 263. The method of any of embodiments 255-257, wherein theviscosity is reduced by 1-10 cP at 25° C.

Embodiment 264. The method of any of embodiments 255-257, wherein theviscosity is reduced by 1-5 cP at 25° C.

Embodiment 265. A method of reducing both the viscosity and theturbidity of an antisense oligonucleotide solution, said methodcomprising adding an effective amount of an excipient that modulatesviscosity, turbidity or both to the antisense oligonucleotide solution.

Embodiment 266. The method of embodiment 265, wherein the excipient thatmodulates viscosity, turbidity or both viscosity and turbidity is anexcipient according to any of embodiments 49-155.

Embodiment 267. The method of embodiment 245 or 246, wherein theantisense compound is an antisense compound according to any ofembodiments 184-234.

Embodiment 268. A method of selecting a pharmaceutically acceptableantisense oligonucleotide from among many antisense oligonucleotides,said method comprising:

-   -   measuring the dynamic viscosity of an antisense oligonucleotide        solution, wherein the solution comprises between 100 mg/mL and        250 mg/mL of the antisense oligonucleotide;    -   measuring the turbidity of the antisense oligonucleotide        solution; and    -   selecting an antisense oligonucleotide that has a dynamic        viscosity below 40 cP and a turbidity below 30 NTU.

Embodiment 269. A method of reducing the turbidity of an antisenseoligonucleotide solution, said method comprising adding an effectiveamount of an excipient that modulates viscosity, turbidity or bothviscosity and turbidity to the antisense oligonucleotide solution.

Embodiment 270. The method of embodiment 269, wherein the excipient thatmodulates viscosity, turbidity or both viscosity and turbidity is anexcipient according to any preceeding embodiment.

Embodiment 271. A method of reducing the viscosity of an antisenseoligonucleotide solution, said method comprising adding an effectiveamount of an excipient that modulates viscosity, turbidity or bothviscosity and turbidity to the antisense oligonucleotide solution.

Embodiment 272. The method of embodiment 271, wherein the excipient thatmodulates viscosity, turbidity or both viscosity and turbidity is anexcipient according to any preceeding embodiment.

Embodiment 273. A method of reducing both the viscosity and theturbidity of an antisense oligonucleotide solution, said methodcomprising adding an effective amount of an excipient that modulatesviscosity, turbidity or both to the antisense oligonucleotide solution.

Embodiment 274. The method of embodiment 273, wherein the excipient thatmodulates viscosity, turbidity or both viscosity and turbidity is anexcipient according to any preceeding embodiment.

Embodiment 275. A method of increasing the viscosity, turbidity, or boththe viscosity and turbidity of an antisense oligonucleotide solution,comprising reducing or removing the amount of an excipient thatmodulates viscosity, turbidity or both from the antisenseoligonucleotide solution.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a standard curve conversion for 13-mm sample tubesfor 0-1000 NTU. CF for the tubes is shown to be the slope of the plot,which is 1.8148.

FIG. 2 illustrates the turbidity and temperature profiles of 220 mg/mLISIS NO. 426115 over time. The turbidity reaches 20 NTU, which isapproximately the lower limit of visible turbidity, at around 50minutes.

FIG. 3 illustrates the turbidity plotted over (approximate) temperatureprofile of 220 mg/mL ISIS NO. 426115

FIGS. 4A and 4B illustrate the effect of NTU of Formazin referencesuspensions on visible turbidity. FIG. 4A These standards are 0(visually clear), 20, 100, 1000, and 4000 (visually turbid) NTU,respectively. FIG. 4B These standards shown in 2 mL, 13 mm vials are 20,10, and 0 (WFI control) NTU respectively.

DETAILED DESCRIPTION OF THE INVENTION

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “aqueous solution” means a solution comprising one ormore solutes in water. In certain embodiments, the water is sterilewater. In certain embodiments, an aqueous solution is saline.

As used herein, “antisense oligonucleotide solution” means an aqueoussolution comprising one or more antisense oligonucleotides. In certainembodiments, an antisense oligonucleotide solution comprises an aqueoussolution having only one antisense oligonucleotide. In certainembodiments, an antisense oligonucleotide solution comprises an aqueoussolution having more than one antisense oligonucleotide. In certainembodiments, an antisense oligonucleotide solution comprises an aqueoussolution wherein an antisense oligonucleotide is present at aconcentration between 0.1 and 500 mg/mL.

As used herein, “excipient” means any compound or composition other thanwater or an antisense oligonucleotide.

As used herein, “excipient that modulates viscosity, turbidity or bothviscosity and turbidity” or “excipient that modulates viscosity,turbidity or both” means an excipient, the presence of which increasesor decreases the viscosity and/or turbidity of an antisenseoligonucleotide solution, compared to the viscosity or turbidity of theantisense oligonucleotide solution at the same concentration andtemperature in the absence of the excipient.

As used herein, “aromatic compound” refers to a mono- or polycycliccarbocyclic ring system having one or more aromatic rings. Preferredaromatic ring systems have from about 5 to about 20 carbon atoms in oneor more rings. Aromatic groups as used herein may optionally includefurther substituent groups.

The term “heterocyclic compound” as used herein, refers to a mono-, orpoly-cyclic ring system that includes at least one heteroatom and isunsaturated, partially saturated, or fully saturated, thereby includingheteroaryl groups and heterocyclic aromatic compounds. Heterocycliccompound is also meant to include fused ring systems wherein one or moreof the fused rings contain at least one heteroatom and the other ringscan contain one or more heteroatoms or optionally contain noheteroatoms. A heterocyclic compound typically includes at least oneatom selected from sulfur, nitrogen or oxygen. In certain embodiments, aheterocyclic compound may include one or more rings, wherein each ringhas one or more heteroatoms. In certain embodiments, a heterocycliccompound includes a monocyclic ring system with one or more heteroatoms.In certain embodiments, a heterocyclic compound includes a monocyclicring system with two or more heteroatoms. Examples of heterocycliccompounds include, but are not limited to, [1,3]dioxolane, pyrrolidine,pyrazoline, pyrazolidine, imidazoline, imidazolidine, piperidine,piperazine, oxazolidine, isoxazolidine, morpholine, thiazolidine,isothiazolidine, quinoxaline, pyridazinone, tetrahydrofuran and thelike. Heterocyclic compounds as used herein may optionally includefurther substituent groups.

As used herein, “heterocyclic aromatic compound” means any compoundcomprising a mono- or poly-cyclic aromatic ring system or fused ringsystem wherein at least one of the rings is aromatic and includes one ormore heteroatoms. Heterocyclic aromatic compounds also include fusedring systems, including systems where one or more of the fused ringscontain no heteroatoms. Heterocyclic aromatic compounds typicallyinclude one ring atom selected from sulfur, nitrogen or oxygen. Examplesof heterocyclic aromatic compounds groups include without limitation,pyridine, pyrazine, pyrimidine, pyrrole, pyrazole, imidazole, thiazole,oxazole, isoxazole, thiadiazole, oxadiazole, thiophene, furan,quinoline, isoquinoline, benzimidazole, benzooxazole, quinoxaline andthe like. Heterocyclic aromatic compounds can be attached to a parentmolecule directly or through a linking moiety such as an aliphatic groupor hetero atom. Heterocyclic aromatic compounds as used herein mayoptionally include further substituent groups.

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Inreference to an oligonucleotide, chemical modification does not includedifferences only in nucleobase sequence. Chemical modifications ofoligonucleotides include nucleoside modifications (including sugarmoiety modifications and nucleobase modifications) and internucleosidelinkage modifications.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugarmoiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl comprisingat least one substituent group that differs from that of a naturallyoccurring sugar moiety. Substituted sugar moieties include, but are notlimited to furanosyl comprising substituents at the 2′-position, the3′-position, the 5′-position and/or the 4′-position.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside is capable of (1) incorporation into an oligonucleotide and(2) hybridization to a complementary nucleoside. Such structures includerings comprising a different number of atoms than furanosyl (e.g., 4, 6,or 7-membered rings); replacement of the oxygen of a furanosyl with anon-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change inthe number of atoms and a replacement of the oxygen. Such structures mayalso comprise substitutions corresponding to those described forsubstituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugarsurrogates optionally comprising additional substituents). Sugarsurrogates also include more complex sugar replacements (e.g., thenon-ring systems of peptide nucleic acid). Sugar surrogates includewithout limitation morpholino, modified morpholinos, cyclohexenyls andcyclohexitols.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified.

As used herein, “heterocyclic base” or “heterocyclic nucleobase” means anucleobase comprising a heterocyclic structure.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” means an atom or group of atoms bound to anoligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid.

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measureable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenlyation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound hybridizes.

As used herein, “mRNA” means an RNA molecule that encodes a protein.

As used herein, “pre-mRNA” means an RNA transcript that has not beenfully processed into mRNA. Pre-RNA includes one or more intron.

As used herein, “transcript” means an RNA molecule transcribed from DNA.Transcripts include, but are not limited to mRNA, pre-mRNA, andpartially processed RNA.

As used herein, “targeting” or “targeted to” means the association of anantisense compound to a particular target nucleic acid molecule or aparticular region of a target nucleic acid molecule. An antisensecompound targets a target nucleic acid if it is sufficientlycomplementary to the target nucleic acid to allow hybridization underphysiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity under stringent conditions. Complementary oligomericcompounds need not have nucleobase complementarity at each nucleoside.Rather, some mismatches are tolerated. In certain embodiments,complementary oligomeric compounds or regions are complementary at 70%of the nucleobases (70% complementary). In certain embodiments,complementary oligomeric compounds or regions are 80% complementary. Incertain embodiments, complementary oligomeric compounds or regions are90% complementary. In certain embodiments, complementary oligomericcompounds or regions are 95% complementary. In certain embodiments,complementary oligomeric compounds or regions are 100% complementary.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an antisense oligonucleotide specifically hybridizes tomore than one target site.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “motif” means a pattern of chemical modifications in anoligomeric compound or a region thereof. Motifs may be defined bymodifications at certain nucleosides and/or at certain linking groups ofan oligomeric compound.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligomeric compound or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligomeric compound or region thereof. The nucleosides of such anoligomeric compound may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleoside have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substuent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentinvention have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms differs from the atomor a group of atoms normally present in the named functional group. Incertain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino(═NR_(bb)), amido (—C(O)N(R_(bb))(R_(cc)) or—N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))—(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonimidoyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S—(O)₂R_(bb)).Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, anoptionally linked chemical functional group or a further substituentgroup with a preferred list including without limitation, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl. Selected substituentswithin the compounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and mean a mono- or polycyclic carbocyclic ringsystem radicals having one or more aromatic rings. Examples of arylgroups include without limitation, phenyl, naphthyl, tetrahydronaphthyl,indanyl, idenyl and the like. Preferred aryl ring systems have fromabout 5 to about 20 carbon atoms in one or more rings. Aryl groups asused herein may optionally include further substituent groups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” mean a radical comprising a mono- orpoly-cyclic aromatic ring, ring system or fused ring system wherein atleast one of the rings is aromatic and includes one or more heteroatoms.Heteroaryl is also meant to include fused ring systems including systemswhere one or more of the fused rings contain no heteroatoms. Heteroarylgroups typically include one ring atom selected from sulfur, nitrogen oroxygen. Examples of heteroaryl groups include without limitation,pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl,furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl,quinoxalinyl and the like. Heteroaryl radicals can be attached to aparent molecule directly or through a linking moiety such as analiphatic group or hetero atom. Heteroaryl groups as used herein mayoptionally include further substituent groups.

Oligomeric Compounds

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, such oligomeric compounds compriseoligonucleotides optionally comprising one or more conjugate and/orterminal groups. In certain embodiments, an oligomeric compound consistsof an oligonucleotide. In certain embodiments, oligonucleotides compriseone or more chemical modifications. Such chemical modifications includemodifications one or more nucleoside (including modifications to thesugar moiety and/or the nucleobase) and/or modifications to one or moreinternucleoside linkage.

Certain Sugar Moieties

In certain embodiments, oligomeric compounds of the invention compriseone or more modified nucleosides comprising a modified sugar moiety.Such oligomeric compounds comprising one or more sugar-modifiednucleosides may have desirable properties, such as enhanced nucleasestability or increased binding affinity with a target nucleic acidrelative to oligomeric compounds comprising only nucleosides comprisingnaturally occurring sugar moieties. In certain embodiments, modifiedsugar moieties are substituted sugar moieties. In certain embodiments,modified sugar moieties are bicyclic or tricyclic sugar moieties. Incertain embodiments, modified sugar moieties are sugar surrogates. Suchsugar surrogates may comprise one or more substitutions corresponding tothose of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more substituent, including but not limitedto substituents at the 2′ and/or 5′ positions. Examples of sugarsubstituents suitable for the 2′-position, include, but are not limitedto: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE”). Incertain embodiments, sugar substituents at the 2′ position is selectedfrom allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, O—C₁-C₁₀substituted alkyl; O—C₁-C₁₀ alkoxy; O—C₁-C₁₀ substituted alkoxy, OCF₃,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where eachRm and Rn is, independently, H or substituted or unsubstituted C₁-C₁₀alkyl. Examples of sugar substituents at the 5′-position, include, butare not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. Incertain embodiments, substituted sugars comprise more than onenon-bridging sugar substituent, for example, 2′-F-5′-methyl sugarmoieties (see, e.g., PCT International Application WO 2008/101157, foradditional 5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, O—C₁-C₁₀ alkoxy; O—C₁-C₁₀ substituted alkoxy, SH, CN, OCN,CF₃, OCF₃, O-alkyl, S-alkyl, N(R_(m))-alkyl; O-alkenyl, S-alkenyl, orN(R_(m))-alkenyl; O-alkynyl, S-alkynyl, N(R_(m))-alkynyl;O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl,O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)),where each R_(m) and R_(n) is, independently, H, an amino protectinggroup or substituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituentgroups can be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b)—)N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′,4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, andanalogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15,2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g.,WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, e.g., WO2008/150729, published Dec. 11, 2008);4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004);4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH2—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surrogates comprise a 4′-sulfur atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran. Such tetrahydropyrans may befurther modified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systemsare known in the art that can be used to modify nucleosides (see, e.g.,review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002,10, 841-854).

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following structure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

Certain Nucleobases

In certain embodiments, nucleosides of the present invention compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present invention comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

Certain Internucleoside Linkages

In certain embodiments, the present invention provides oligomericcompounds comprising linked nucleosides. In such embodiments,nucleosides may be linked together using any internucleoside linkage.The two main classes of internucleoside linking groups are defined bythe presence or absence of a phosphorus atom. Representative phosphoruscontaining internucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of the oligomericcompound. In certain embodiments, internucleoside linkages having achiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Certain Motifs

In certain embodiments, the present invention provides oligomericcompounds comprising oligonucleotides. In certain embodiments, sucholigonucleotides comprise one or more chemical modification. In certainembodiments, chemically modified oligonucleotides comprise one or moremodified nucleosides. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleosides comprisingmodified sugars. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleosides comprisingone or more modified nucleobases. In certain embodiments, chemicallymodified oligonucleotides comprise one or more modified internucleosidelinkages. In certain embodiments, the chemically modifications (sugarmodifications, nucleobase modifications, and/or linkage modifications)define a pattern or motif. In certain embodiments, the patterns ofchemical modifications of sugar moieties, internucleoside linkages, andnucleobases are each independent of one another. Thus, anoligonucleotide may be described by its sugar modification motif,internucleoside linkage motif and/or nucleobase modification motif (asused herein, nucleobase modification motif describes the chemicalmodifications to the nucleobases independent of the sequence ofnucleobases).

Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar modification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar modification motif, which comprises twoexternal regions or “wings” and an internal region or “gap.” The threeregions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar modification motifs of the two wings are the sameas one another (symmetric gapmer). In certain embodiments, the sugarmodification motifs of the 5′-wing differs from the sugar modificationmotif of the 3′-wing (asymmetric gapmer). In certain embodiments,oligonucleotides comprise 2′-MOE modified nucleosides in the wings and2′-F modified nucleosides in the gap.

In certain embodiments, oligonucleotides are fully modified. In certainsuch embodiments, oligonucleotides are uniformly modified. In certainembodiments, oligonucleotides are uniform 2′-MOE. In certainembodiments, oligonucleotides are uniform 2′-F. In certain embodiments,oligonucleotides are uniform morpholino. In certain embodiments,oligonucleotides are uniform BNA. In certain embodiments,oligonucleotides are uniform LNA. In certain embodiments,oligonucleotides are uniform cEt.

In certain embodiments, oligonucleotides comprise a uniformly modifiedregion and additional nucleosides that are unmodified or differentlymodified. In certain embodiments, the uniformly modified region is atleast 5, 10, 15, or 20 nucleosides in length. In certain embodiments,the uniform region is a 2′-MOE region. In certain embodiments, theuniform region is a 2′-F region. In certain embodiments, the uniformregion is a morpholino region. In certain embodiments, the uniformregion is a BNA region. In certain embodiments, the uniform region is aLNA region. In certain embodiments, the uniform region is a cEt region.

In certain embodiments, the oligonucleotide does not comprise more than4 contiguous unmodified 2′-deoxynucleosides. In certain circumstances,antisesense oligonucleotides comprising more than 4 contiguous2′-deoxynucleosides activate RNase H, resulting in cleavage of thetarget RNA. In certain embodiments, such cleavage is avoided by nothaving more than 4 contiguous 2′-deoxynucleosides, for example, wherealteration of splicing and not cleavage of a target RNA is desired.

Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif, as described above for sugar modification motif. In suchembodiments, the internucleoside linkages in each of two wing regionsare different from the internucleoside linkages in the gap region. Incertain embodiments the internucleoside linkages in the wings arephosphodiester and the internucleoside linkages in the gap arephosphorothioate. The sugar modification motif is independentlyselected, so such oligonucleotides having a gapped internucleosidelinkage motif may or may not have a gapped sugar modification motif andif it does have a gapped sugar motif, the wing and gap lengths may ormay not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present invention comprise a region of uniformlymodified internucleoside linkages. In certain such embodiments, theoligonucleotide comprises a region that is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide is uniformly linked by phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate and at least oneinternucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certain suchembodiments, nucleobase modifications are arranged in a gapped motif. Incertain embodiments, nucleobase modifications are arranged in analternating motif. In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

Certain Overall Lengths

In certain embodiments, the present invention provides oligomericcompounds including oligonucleotides of any of a variety of ranges oflengths. In certain embodiments, the invention provides oligomericcompounds or oligonucleotides consisting of X to Y linked nucleosides,where X represents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≤Y. For example, in certainembodiments, the invention provides oligomeric compounds which compriseoligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linkednucleosides. In embodiments where the number of nucleosides of anoligomeric compound or oligonucleotide is limited, whether to a range orto a specific number, the oligomeric compound or oligonucleotide may,nonetheless further comprise additional other substituents. For example,an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotideshaving 31 nucleosides, but, unless otherwise indicated, such anoligonucleotide may further comprise, for example one or moreconjugates, terminal groups, or other substituents. In certainembodiments, a gapmer oligonucleotide has any of the above lengths.

One of skill in the art will appreciate that certain lengths may not bepossible for certain motifs. For example: a gapmer having a 5′-wingregion consisting of four nucleotides, a gap consisting of at least sixnucleotides, and a 3′-wing region consisting of three nucleotides cannothave an overall length less than 13 nucleotides. Thus, one wouldunderstand that the lower length limit is 13 and that the limit of 10 in“10-20” has no effect in that embodiment.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range. For example, an oligonucleotide consisting of20-25 linked nucleosides comprising a 5′-wing consisting of 5 linkednucleosides; a 3′-wing consisting of 5 linked nucleosides and a centralgap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap(before reaching the overall length limitation of 25). Such additionalnucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing.

Certain Oligonucleotides

In certain embodiments, oligonucleotides of the present invention arecharacterized by their sugar motif, internucleoside linkage motif,nucleobase modification motif and overall length. In certainembodiments, such parameters are each independent of one another. Thus,each internucleoside linkage of an oligonucleotide having a gapmer sugarmotif may be modified or unmodified and may or may not follow the gapmermodification pattern of the sugar modifications. Thus, theinternucleoside linkages within the wing regions of a sugar-gapmer maybe the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region. Likewise,such sugar-gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.Herein if a description of an oligonucleotide or oligomeric compound issilent with respect to one or more parameter, such parameter is notlimited. Thus, an oligomeric compound described only as having a gapmersugar motif without further description may have any length,internucleoside linkage motif, and nucleobase modification motif. Unlessotherwise indicated, all chemical modifications are independent ofnucleobase sequence.

Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamics, pharmacokinetics, stability, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligomericcompound, such as an oligonucleotide. Conjugate groups includes withoutlimitation, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groupshave been described previously, for example: cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

In certain embodiments, conjugate groups are directly attached tooligonucleotides in oligomeric compounds. In certain embodiments,conjugate groups are attached to oligonucleotides by a conjugate linkinggroup. In certain such embodiments, conjugate linking groups, including,but not limited to, bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Conjugate linkinggroups are useful for attachment of conjugate groups, such as chemicalstabilizing groups, functional groups, reporter groups and other groupsto selective sites in a parent compound such as for example anoligomeric compound. In general a bifunctional linking moiety comprisesa hydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such aschemical functional group or a conjugate group. In some embodiments, theconjugate linker comprises a chain structure or an oligomer of repeatingunits such as ethylene glycol or amino acid units. Examples offunctional groups that are routinely used in a bifunctional linkingmoiety include, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties includepyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′ end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group.

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, oligomeric compounds comprise anoligonucleotide. In certain embodiments, an oligomeric compoundcomprises an oligonucleotide and one or more conjugate and/or terminalgroups. Such conjugate and/or terminal groups may be added tooligonucleotides having any of the chemical motifs discussed above.Thus, for example, an oligomeric compound comprising an oligonucleotidehaving region of alternating nucleosides may comprise a terminal group.Antisense Compounds

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. Such antisense compounds are capable ofhybridizing to a target nucleic acid, resulting in at least oneantisense activity. In certain embodiments, antisense compoundsspecifically hybridize to one or more target nucleic acid. In certainembodiments, a specifically hybridizing antisense compound has anucleobase sequence comprising a region having sufficientcomplementarity to a target nucleic acid to allow hybridization andresult in antisense activity and insufficient complementarity to anynon-target so as to avoid non-specific hybridization to any non-targetnucleic acid sequences under conditions in which specific hybridizationis desired (e.g., under physiological conditions for in vivo ortherapeutic uses, and under conditions in which assays are performed inthe case of in vitro assays).

In certain embodiments, the present invention provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

TABLE 1 below provides certain non-limiting examples of antisensecompounds and their targets:

TABLE 1 Antisense Compounds Target ISIS No Indication Sequence ChemistrySEQ ID NO Factor XI 416858 Clotting ACGGCATTGGTGCACAGTTT 5-10-5 MOE  1disorder TTR 420915 Amyloides TCTTGGTTACATGAAATCCC 5-10-5 MOE  2 Apo(a)494372 CAD TGCTCCGTTGGTGCTTGTTC 5-10-5 MOE  3 Alpha1- 487660Liver disease CCAGCTCAACCCTTCTTTAA 5-10-5 MOE  4 antitrypsin PTP-1B404173 Diabetes AATGGTTTATTCCATGGCCA 5-10-5 MOE  5 GCGR 449884 DiabetesGGTTCCCGAGGTGCCCA 3-10-4 MOE  6 DGAT2 501861 NASH TCACAGAATTATCAGCAGTA5-10-5 MOE  7 Factor VII 540175 Cancer- GGACACCCACGCCCCC 3-10-3  8associated cEt/MOE thrombosis SMN 396443 SMA TCACTTTCATAATGCTGG Umiform 9 MOE FGFR4 463588 Obesity GCACACTCAGCAGGACCCCC 5-10-5 MOE 10 apoB-100301012 High GCCTCAGTCTGCTTCGCACC 5-10-5 MOE 11 Cholesterol CRP 329993CAD AGCATAGTTAACGAGCTCCC 5-10-5 MOE 12 ApoC-III 304801 HighAGCTTCTTGTCCAGCTTTAT 5-10-5 MOE 13 triglycerides GCCR 426115 DiabetesGCAGCCATGGTGATCAGGAG 5-10-5 MOE 14 STAT3 481464 Cancer CTATTTGGATGTCAGC3-10-3 (S)-cEt 15 eIF-4E 183750 Cancer TGTCATATTCCTGGATCCTT 5-10-5 MOE16 SOD1 333611 ALS CCGTCGCCCTTCAGCACGCA 5-10-5 MOE 17 GHR 227452Acromegaly TCAGGGCATTCTTTCCATTC 5-10-5 MOE 18 Clusterin 112989 CancerCAGCAGCAGAGTCTTCATCAT 4-13-4 MOE 19 Hsp27 306053 CancerGGGACGCGGCGCTCGGTCAT 4-12-4 MOE 20 CMV   2922 RetinitisGCGTTTGCTCTTCTTCTTGCG Uniform deoxy 21 ICAM-1   2302 UlcerativeGCCCAAGCTGGCATCCGTCA Uniform deoxy 22 colitis VLA-4 107248 MultipleCTGAGTCTGTTTTCCATTCT 3-9-8 MOE 23 sclerosis CTGF 412294 FibrosisGTTTGACATGGCACAATGTT 2-13-5 MOE 24 c-raf kinase  13650 Ocular diseaseTCCCGCCTGTGACATGCATT 6-8-6 MOE 25Certain Target Nucleic Acids and Mechanisms

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid. In certain embodiments, the target nucleic acid is anendogenous RNA molecule. In certain embodiments, the target nucleic acidis a pre-mRNA. In certain embodiments, an antisense oligonucleotidemodulates splicing of a pre-mRNA.

Certain Properties of Antisense Compounds

Viscosity

In certain embodiments, antisense oligonucleotide solutions possessvarying degrees of viscosity, and the viscosity of antisenseoligonucleotide solutions depends on many factors. Some factors include,but are not limited to, length, nucleobase sequence, nucleobasemodifications, nucleobase modification motif, and/or sugar modificationof the antisense oligonucleotide. In certain embodiments, the viscosityof an antisense oligonucleotide solution may be difficult to predict. Incertain embodiments an antisense oligonucleotide solution may exhibit arelatively high viscosity, while an antisense oligonucleotide solutioncomprising a similar antisense oligonucleotide (partially homologoussequence, similar modifications, etc.) may exhibit a relatively lowviscosity. In certain embodiments it may be desireable to lower theviscosity of a given antisense oligonucleotide solution, for example, incertain embodiments it may be desirable to to lower the viscosity of agiven antisense oligonucleotide solution so that higher amounts of anantisense oligonucleotide may be present in a given volume of solution.

In certain embodiments it may be desirable to increase the viscosity ofa given antisense oligonucleotide solution. In certain embodiments,viscosity may be increased or decreased depending on a variety offactors, e.g. concentration, volume of solute, temperature, and/or pH.In certain embodiments, the desireable viscosity varies. In certainembodiments it may be desirable to increase or decrease viscosity,depending on the particular route of delivery or application a givenantisense oligonucleotide solution.

In certain embodiments, viscosity is often a concentration-limitingfactor. While not wishing to be bound by theory, in certain embodiments,as the concentration of an antisense oligonucleotide is increased,interactions between antisense oligonucleotide molecules likewiseincrease. In certain embodiments, certain antisense oligonucleotides mayinteract to form aggregates which may increase viscosity and/orturbidity. Certain antisense oligonucleotides may interact to formantisense oligonucleotide polymers, which may increase viscosity and/orturbidity. In certain embodiments, as the concentration of an antisenseoligonucleotide in solution increases, the viscosity of the antisenseoligonucleotide solution also increases. In certain embodiments, it isundesirable to have an antisense oligonucleotide solution having a highviscosity. For example, if the viscosity of an antisense oligonucleotidesolution is too high, then it may become difficult to manufacture anantisense oligonucleotide solution including but not limited to anantisense oligonucleotide solution drug product. As another example, ifthe viscosity of an antisense oligonucleotide solution is too high,filtration time during manufacturing may increase, adding time and costto the manufacturing process. As another example, if the viscosity of anantisense oligonucleotide solution is too high, it may become moredifficult to pre-fill syringes with accurate and precise amounts of theantisense oligonucleotide solution. As another example, if the viscosityof an antisense oligonucleotide solution is too high, it may become moredifficult to administer to an animal, human, or patient. As anotherexample, if the viscosity of an antisense oligonucleotide solution istoo high, drug clearance from the subcutaneous injection site may beslowed. As another example, if the viscosity of an antisenseoligonucleotide solution is too high, a larger gauge needle may berequired to effectively administer doses of the antisenseoligonucleotide solution, which may exacerbate discomfort uponinjection. In certain embodiments, it is therefore desirable to have anantisense oligonucleotide solution having both a high concentration ofantisense oligonucleotide and low viscosity.

In certain embodiments, if the viscosity of an antisense oligonucleotidesolution is too high, then the concentration of the antisenseoligonucleotide in solution may be reduced to reduce the viscosity ofthe antisense oligonucleotide solution to a desirable level. Thereduction of the concentration of an antisense oligonucleotide in theantisense oligonucleotide solution may be undesirable for severalreasons. For example, as the concentration of an antisenseoligonucleotide in an antisense oligonucleotide solution is decreased,animals, humans, and/or patients must receive a larger volume ofantisense oligonucleotide solution to receive the desired amount of anantisense oligonucleotide. In certain embodiments, it is thereforedesirable to reduce the viscosity of an antisense oligonucleotidesolution without reducing the concentration of the antisenseoligonucleotide within the antisense oligonucleotide solution. Incertain embodiments, it is therefore desirable to have an antisenseoligonucleotide solution having both a high concentration of antisenseoligonucleotide and low viscosity.

In certain embodiments, certain antisense oligonucleotide solutionshaving desirable concentrations of antisense oligonucleotides haveundesirably high viscosities. In certain embodiments, the addition ofone or more excipients that modulate viscosity, turbidity or both mayreduce the viscosity of a certain antisense oligonucleotide solution toa desirable level.

In certain embodiments described herein, it is recognized that viscosityof an oligonucleotide solution may vary with temperature. In certainembodiments, the viscosity may be expressed as a range of cP unitsrelative to a concentration of oligonucleotide in solution. For example,in certain embodiments, the viscosity of the antisense oligonucleotidesolution is less than 40 cP when the concentration of the antisenseoligonucleotide is between 40 to 60 mg/mL. In certain such embodiments,the viscocity of the solution and the concentration of theoligonucleotide represent an approximate measurement of viscocity andapproximate concentration of oligonucleotide at a given temperature orrange of temperatures. For example, in certain embodiments describedherein, the temperature of the solution for which viscocity is measuredis about 25 degrees Celsius.

In certain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution reduces the viscosity of the antisense oligonucleotide solutionto less than 40 cP when the concentration of the antisenseoligonucleotide is between 40 to 60 mg/mL. In certain embodiments, theaddition of one or more excipients that modulate viscosity, turbidity orboth to the antisense oligonucleotide solution reduces the viscosity ofthe antisense oligonucleotide solution to less than 40 cP when theconcentration of the antisense oligonucleotide is between 45 to 55mg/mL. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the viscosity of the antisenseoligonucleotide solution to less than 40 cP when the concentration ofthe antisense oligonucleotide is between 80 to 120 mg/mL. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionreduces the viscosity of the antisense oligonucleotide solution to lessthan 40 cP when the concentration of the antisense oligonucleotide isbetween 90 to 110 mg/mL. In certain embodiments, the addition of one ormore excipients that modulate viscosity, turbidity or both to theantisense oligonucleotide solution reduces the viscosity of theantisense oligonucleotide solution to less than 40 cP when theconcentration of the antisense oligonucleotide is between 140 to 160mg/mL. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the viscosity of the antisenseoligonucleotide solution to less than 40 cP when the concentration ofthe antisense oligonucleotide is between 165 to 185 mg/mL. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionreduces the viscosity of the antisense oligonucleotide solution to lessthan 40 cP when the concentration of the antisense oligonucleotide isbetween 180 to 220 mg/mL. In certain embodiments, the addition of one ormore excipients that modulate viscosity, turbidity or both to theantisense oligonucleotide solution reduces the viscosity of theantisense oligonucleotide solution to less than 40 cP when theconcentration of the antisense oligonucleotide is between 190 to 210mg/mL. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the viscosity of the antisenseoligonucleotide solution to less than 40 cP when the concentration ofthe antisense oligonucleotide is between 210 to 230 mg/mL. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionreduces the viscosity of the antisense oligonucleotide solution to lessthan 40 cP when the concentration of the antisense oligonucleotide isbetween 230 to 260 mg/mL. In certain embodiments, the addition of one ormore excipients that modulate viscosity, turbidity or both to theantisense oligonucleotide solution reduces the viscosity of theantisense oligonucleotide solution to less than 40 cP when theconcentration of the antisense oligonucleotide is between 245 to 255mg/mL. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the viscosity of the antisenseoligonucleotide solution to less than 40 cP when the concentration ofthe antisense oligonucleotide is between 260 to 300 mg/mL. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionreduces the viscosity of the antisense oligonucleotide solution to lessthan 40 cP when the concentration of the antisense oligonucleotide isbetween 300 to 400 mg/mL.

In certain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution reduces the viscosity of the antisense oligonucleotide solutionto less than 40 cP when the antisense oligonucleotide has one or moremodified sugars having 2′-MOE modifications. In certain embodiments, theaddition of one or more excipients that modulate viscosity, turbidity orboth to the antisense oligonucleotide solution reduces the viscosity ofthe antisense oligonucleotide solution to less than 40 cP when theantisense oligonucleotide has one or more modified sugars having 2′-OMemodifications. In certain embodiments, the addition of one or moreexcipients that modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the viscosity of the antisenseoligonucleotide solution to less than 40 cP when the antisenseoligonucleotide has one or more modified sugars having 2′-Fmodifications. In certain embodiments, the addition of one or moreexcipients that modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the viscosity of the antisenseoligonucleotide solution to less than 40 cP when the antisenseoligonucleotide has one or more modified sugars having LNAmodifications. In certain embodiments, the addition of one or moreexcipients that modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the viscosity of the antisenseoligonucleotide solution to less than 40 cP when the antisenseoligonucleotide has one or more modified sugars having cEtmodifications.

Turbidity

While not wishing to be bound by theory, the presence of turbidity inantisense oligonucleotide solutions is associated with the formation ofantisense oligonucleotide strand aggregates. In certain embodiments, thepresence of turbidity in antisense oligonucleotide solutions makes theantisense oligonucleotide solution appear white and cloudy. In certainembodiments, the presence of turbidity in antisense oligonucleotidesolutions makes the antisense oligonucleotide solution appear to containsmall particles. In certain embodiments, it is desirable to haveantisense oligonucleotide solutions that have low turbidity. In certainembodiments, it is desirable to have antisense oligonucleotide solutionsthat appear clear and particle-free when viewed by the naked eye. Incertain embodiments, it is desirable to have antisense oligonucleotidesolutions that have turbidity below 20 NTU.

In certain embodiments, turbidiy may be induced via a freeze-thawmethod, wherein an antisense oligonucleotide solution is frozen and thenrapidly thawed, as described herein. In certain embodiments, thefreese-thaw method induces turbidity wherein a given antisenseoligonucleotide solution would not normally demonstrate turbidity, orwherein a given antisense oligonucleotide solution would demonstrateturbidity after a long period of time. In certain embodiments turbidityrepresents an aesthetic problem, but does not affect the efficacy orsafety of a given antisense oligonucleotide solution.

In certain embodiments, certain antisense oligonucleotide solutionshaving desirable concentrations of antisense oligonucleotides haveundesirably high turbidity. In certain embodiments, certain antisenseoligonucleotide solutions having desirable concentrations of antisenseoligonucleotides have undesirably high turbidity wherein the antisenseoligonucleotide solution has a cloudy or milky white appearance. Incertain embodiments, certain antisense oligonucleotide solutions havingdesirable concentrations of antisense oligonucleotides have undesirablyhigh turbidity wherein the antisense oligonucleotide solution appears tohave small amounts of particulate interspersed throughout the antisenseoligonucleotide solution. In certain embodiments, the addition of one ormore excipients that modulate viscosity, turbidity or both may reducethe turbidity of a certain antisense oligonucleotide solution to adesirable level. In certain embodiments, the addition of one or moreexcipients that modulate viscosity, turbidity or both may reduce theturbidity of a certain antisense oligonucleotide solution from having acloudy appearance to a having a clear appearance to the naked eye. Incertain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both may reduce the turbidity of acertain antisense oligonucleotide solution from having a milky whiteappearance to a having a clear appearance to the naked eye. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both may reduce the turbidity of a certainantisense oligonucleotide solution from visible particles to a having aclear particle-free appearance to the naked eye.

In certain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution reduces the turbidity of the antisense oligonucleotide solutionto less than 20 NTU when the concentration of the antisenseoligonucleotide is between 40 to 60 mg/mL. In certain embodiments, theaddition of one or more excipients that modulate viscosity, turbidity orboth to the antisense oligonucleotide solution reduces the turbidity ofthe antisense oligonucleotide solution to less than 20 NTU when theconcentration of the antisense oligonucleotide is between 45 to 55mg/mL. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the turbidity of the antisenseoligonucleotide solution to less than 20 NTU when the concentration ofthe antisense oligonucleotide is between 80 to 120 mg/mL. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionreduces the turbidity of the antisense oligonucleotide solution to lessthan 20 NTU when the concentration of the antisense oligonucleotide isbetween 90 to 110 mg/mL. In certain embodiments, the addition of one ormore excipients that modulate viscosity, turbidity or both to theantisense oligonucleotide solution reduces the turbidity of theantisense oligonucleotide solution to less than 20 NTU when theconcentration of the antisense oligonucleotide is between 140 to 160mg/mL. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the turbidity of the antisenseoligonucleotide solution to less than 20 NTU when the concentration ofthe antisense oligonucleotide is between 165 to 185 mg/mL. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionreduces the turbidity of the antisense oligonucleotide solution to lessthan 20 NTU when the concentration of the antisense oligonucleotide isbetween 180 to 220 mg/mL. In certain embodiments, the addition of one ormore excipients that modulate viscosity, turbidity or both to theantisense oligonucleotide solution reduces the turbidity of theantisense oligonucleotide solution to less than 20 NTU when theconcentration of the antisense oligonucleotide is between 190 to 210mg/mL. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the turbidity of the antisenseoligonucleotide solution to less than 20 NTU when the concentration ofthe antisense oligonucleotide is between 210 to 230 mg/mL. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionreduces the turbidity of the antisense oligonucleotide solution to lessthan 20 NTU when the concentration of the antisense oligonucleotide isbetween 230 to 260 mg/mL. In certain embodiments, the addition of one ormore excipients that modulate viscosity, turbidity or both to theantisense oligonucleotide solution reduces the turbidity of theantisense oligonucleotide solution to less than 20 NTU when theconcentration of the antisense oligonucleotide is between 245 to 255mg/mL. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the turbidity of the antisenseoligonucleotide solution to less than 20 NTU when the concentration ofthe antisense oligonucleotide is between 260 to 300 mg/mL. In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionreduces the turbidity of the antisense oligonucleotide solution to lessthan 20 NTU when the concentration of the antisense oligonucleotide isbetween 300 to 400 mg/mL.

In certain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution reduces the turbidity of the antisense oligonucleotide solutionto less than 20 NTU when the antisense oligonucleotide has one or moremodified sugars having 2′-MOE modifications. In certain embodiments, theaddition of one or more excipients that modulate viscosity, turbidity orboth to the antisense oligonucleotide solution reduces the turbidity ofthe antisense oligonucleotide solution to less than 20 NTU when theantisense oligonucleotide has one or more modified sugars having 2′-OMemodifications. In certain embodiments, the addition of one or moreexcipients that modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the turbidity of the antisenseoligonucleotide solution to less than 20 NTU when the antisenseoligonucleotide has one or more modified sugars having 2′-Fmodifications. In certain embodiments, the addition of one or moreexcipients that modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the turbidity of the antisenseoligonucleotide solution to less than 20 NTU when the antisenseoligonucleotide has one or more modified sugars having LNAmodifications. In certain embodiments, the addition of one or moreexcipients that modulate viscosity, turbidity or both to the antisenseoligonucleotide solution reduces the turbidity of the antisenseoligonucleotide solution to less than 20 NTU when the antisenseoligonucleotide has one or more modified sugars having cEtmodifications.

Osmolarity

While not wishing to be bound by theory, antisense oligonucleotidesolutions possess varying concentrations of osmolarity, and theosmolarity of antisense oligonucleotide solutions depends on manyfactors. Some factors include but are not limited to, length, nucleobasesequence, nucleobase modifications, nucleobase modification motif,and/or sugar modification of the antisense oligonucleotide. In certainembodiments, the osmolarity of an antisense oligonucleotide solution maybe difficult to predict. In certain embodiments an antisenseoligonucleotide solution may exhibit a relatively high concentrations ofosmolarity, while a similar antisense oligonucleotide solution mayexhibit a relatively low concentrations of osmolarity.

In certain embodiments, certain antisense oligonucleotide solutionshaving desirable concentrations of antisense oligonucleotides haveundesirably low osmolarity, and are hypotonic. In certain embodiments,it is undesirable to have an antisense oligonucleotide solution havinglow osmolarity, and hypotonicity. For example, if the osmoloarity of anantisense oligonucleotide solution is too low, then an animal, human, orpatient may experience pain at the injection site. In certainembodiments, it is therefore desirable to increase the osmolarity of anantisense oligonucleotide solution. In certain embodiments, it istherefore desirable to have the antisense oligonucleotide solutionbecome isotonic.

In certain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution increases the osmolarity of the antisense oligonucleotidesolution. In certain embodiments, the addition of one or more excipientsthat modulate viscosity, turbidity or both to the antisenseoligonucleotide solution increases the osmolarity of the antisenseoligonucleotide solution wherein the antisense oligonucleotide solutionbecomes isotonic.

pH

While not wishing to be bound by theory, antisense oligonucleotidesolutions possess varying pH levels, and the pH levels of antisenseoligonucleotide solutions depends on many factors. Some factors include,but are not limited to length, nucleobase sequence, nucleobasemodifications, nucleobase modification motif, and/or sugar modificationof the antisense oligonucleotide. In certain embodiments, the pH of anantisense oligonucleotide solution may be difficult to predict. Incertain embodiments an antisense oligonucleotide solution may exhibit arelatively high pH, while an antisense oligonucleotide solution of asimilar antisense oligonucleotide may exhibit a relatively low pH.

In certain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution alters the pH of the antisense oligonucleotide solution. Incertain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution increases pH of the antisense oligonucleotide solution. Incertain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution decreases pH of the antisense oligonucleotide solution. Incertain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution buffers pH of the antisense oligonucleotide solution. Incertain embodiments, the addition of one or more excipients thatmodulate viscosity, turbidity or both to the antisense oligonucleotidesolution buffers the pH of the antisense oligonucleotide solution arounda pH of 8.0. (e.g., 7.8 to 8.2) In certain embodiments, the addition ofone or more excipients that modulate viscosity, turbidity or both to theantisense oligonucleotide solution buffers the pH of the antisenseoligonucleotide solution around a pH of 7.4. (e.g., 7.2-7.6) In certainembodiments, the addition of one or more excipients that modulateviscosity, turbidity or both to the antisense oligonucleotide solutionbuffers the pH of the antisense oligonucleotide solution around a pH of7.0. (e.g. 6.8-7.2).

Antimicrobial

In certain embodiments it is desirable to include an antimicrobial agentto an antisense oligonucleotide solution to facilitate storage ordelivery. In certain embodiments it is not necessary to include anantimicrobial agent to an antisense oligonucleotide solution tofacilitate storage or delivery of the antisense oligonucleotidesolution. In certain embodiments an antimicrobial agent is added to anantisense oligonucleotide solution. In certain embodiments noantimicrobial agents are added to an antisense oligonucleotide solution.In certain embodiments antisense oligonucleotide solutions are preparedwithout antimicrobial agents. In certain embodiments one or moreexcipients that modulate viscosity, turbidity or both may also serve asan antimicrobial agent or preservative. In certain embodiments one ormore excipients that modulate viscosity, turbidity or both may not haveany antimicrobial or preservative properties.

Certain Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active antisense oligomericcompound.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present invention provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human.

In certain embodiments, the present invention provides methods ofadministering a pharmaceutical composition comprising an oligomericcompound of the present invention to an animal. Suitable administrationroutes include, but are not limited to, oral, rectal, transmucosal,intestinal, enteral, topical, suppository, through inhalation,intrathecal, intracerebroventricular, intraperitoneal, intranasal,intraocular, intratumoral, and parenteral (e.g., intravenous,intramuscular, intramedullary, and subcutaneous). In certainembodiments, pharmaceutical intrathecals are administered to achievelocal rather than systemic exposures. For example, pharmaceuticalcompositions may be injected directly in the area of desired effect(e.g., into the eyes, ears).

Certain Excipients

As reported in scientific or patent literature, some oligonucleotidecompounds form aggregates when in solution and aggregation may causesundesired viscosity, turbidity, or both. In certain embodiments,oligonucleotide solutions having high viscosity are undesirable becauseit complicates delivery through a syringe. For example, oligonucleotidesolutions having high viscosity may only effectively get delivered usinga high gauge needle which may cause excess pain or discomfort to apatient. In certain embodiments, oligonucleotide solutions having highturbidity are undesirable because the oligonucleotide solution mayappear to have one or more impurities present.

In certain embodiments, oligonucleotide solutions having additives canmitigate only turbidity or only viscosity (see US 20110098343). Incertain embodiments, this disclosure provides that both turbidity andviscosity can be comitigated by a single excipient, for example,L-tryptophan, pyridoxine, L-Phenylalanine, and nicotinamide. In certainembodiments, additional excipients that have potential to mitigate bothturbidity and viscosity include but are not limited to thymine, adenine,riboflavin, thiamine, and tryptamine. In certain embodiments, effectiveexcipients have heterocyclic character and/or an ability to act as ahydrogen bond donor and/or a hydrogen bond acceptor. In certainembodiments, excipients with nonaromatic rings may not be effective forviscosity reduction.

In certain embodiments, certain properties of effective excipients, e.g.(i) aromatic homocyclicity or heterocyclicity and (ii) the ability toact as a hydrogen bond donor and/or acceptor, may be consolidated intoone excipient or compound. In certain embodiments, two or moreexcipients may together possess one or more properties of effectiveexcipients. For example, in certain embodiments, a first excipient maybe an aromatic homocyclic or heterocyclic compound but may not have theability to act as a hydrogen bond donor and/or acceptor. In certain suchembodiments, a second excipient may have the ability to act as ahydrogen bond donor and/or acceptor, but may not be an aromatichomocyclic or heterocyclic compound. In certain such embodiments, thefirst excipient may act as a aromatic homocyclic or heterocycliccompound and a second excipient may act as a hydrogen bond donor and/oracceptor and the combination of the first and second excipient mayproduce an effective excipient for mitigating turbidity, viscocity, orboth. In certain embodiments, a mixture of two or more excipients, thesum of which contain (i) aromatic homocyclicity or heterocyclicity and(ii) the ability to act as a hydrogen bond donor and/or acceptor, areeffective in mitigating both turbidity and viscosity.

In certain embodiments, antisense oligonucleotide compositions providedherein comprise one or more modified antisense oligonucleotides and oneor more excipients. Any suitable excipient known to those having skillin the art may be used. For example, suitable excipients may be foundin, for example “Handbook of Pharmaceutical Excipients,” AmericanPharmaceutical Association Publications, Washington D.C., 6^(th)Edition, 2009; which is hereby incorporated herein by reference in itsentirety. In certain such embodiments, excipients are selected from saltsolutions, alcohol, polyethylene glycols, gelatin, lactose, amylase,magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, excipients are selected from Adenine, BenzylAlcohol, m-Cresol, Cytidine, Cytidine Monophosphate, Cytosine, Dextran,Guanine Monophosphate, D-Mannitol, Methylparaben, Nicotinamide, Phenol,2-Phenoxyethanol, L-Phenylalanine, Pyridoxine, Sodium Chloride, Thymine,tryptophan, L-Tryptophan, L-Tyrosine, Ascorbic Acid, Benzamide,o-Benzenediol, Benzenehexol, L-Histidine, Hydroxypyridine, Indole,D-Mannitol+Phenol mixture, D-Mannitol+Pyridine mixture, 2H-Pyran,Pyrazinamide, Pyridine, Pyrimidine, 2-Pyrone, Riboflavin, Thiamine,Tryptamine, ethanol, (2-Hydroxypropyl)-β-cyclodextrin, Niacin,Polyethylene Glycol 600, Polyethylene Glycol 4600, Propylene Glycol,Pyridoxine, Sucrose, Thymidine, Tween 80, Uridine, Thymine, caffeine,acridine orange, ethidium bromide, propidium iodide, cyanine dyes suchas PicoGreen®, thiamine hydrochloride, ethylene diamine tetraaceticacid, and 1,2-dihydroxybenzene.

In certain embodiments, excipients comprise heterocyclic molecules. Incertain embodiments, excipients comprise heterocyclic amines. In certainembodiments, excipients comprise aromatic molecules or molecules havingone one or more aromatic ring. In certain embodiments, excipientscomprise heterocyclic molecules wherein the heteroatom is oxygen. Incertain embodiments, excipients comprise heterocyclic molecules whereinthe heteroatom is nitrogen. In certain embodiments, excipients compriseheterocyclic molecules wherein the heteroatom is sulfur.

In certain embodiments, the excipient lowers the viscosity of anantisense oligonucleotide composition. In certain embodiments, theexcipient lowers the turbidity of an antisense oligonucleotidecomposition. In certain embodiments, the excipient increases theosmolarity of an antisense oligonucleotide composition. In certainembodiments, the excipient decreases the osmolarity of an antisenseoligonucleotide composition. In certain embodiments, the excipientincreases the pH of an antisense oligonucleotide composition. In certainembodiments, the excipient decreases the pH of an antisenseoligonucleotide composition. In certain embodiments, the excipientbuffers the pH of an antisense oligonucleotide composition. In certainembodiments, the excipient lowers the turbidity and the viscosity of anantisense oligonucleotide composition. In certain embodiments, theexcipient lowers the turbidity and the viscosity of an antisenseoligonucleotide composition and also increases the osmolarity of theantisense composition. In certain embodiments, the excipient lowers theturbidity and the viscosity of an antisense oligonucleotide compositionand also decreases the osmolarity of the antisense composition.

In certain embodiments, a mixture of two or more excipients lowers theviscosity of an antisense oligonucleotide composition. In certainembodiments, a mixture of two or more excipients lowers the turbidity ofan antisense oligonucleotide composition. In certain embodiments, amixture of two or more excipients increases the osmolarity of anantisense oligonucleotide composition. In certain embodiments, a mixtureof two or more excipients decreases the osmolarity of an antisenseoligonucleotide composition. In certain embodiments, a mixture of two ormore excipients increases the pH of an antisense oligonucleotidecomposition. In certain embodiments, a mixture of two or more excipientsdecreases the pH of an antisense oligonucleotide composition. In certainembodiments, a mixture of two or more excipients buffers the pH of anantisense oligonucleotide composition. In certain embodiments, a mixtureof two or more excipients lowers the turbidity and the viscosity of anantisense oligonucleotide composition. In certain embodiments, a mixtureof two or more excipients lowers the turbidity and the viscosity of anantisense oligonucleotide composition and also increases the osmolarityof the antisense composition. In certain embodiments, a mixture of twoor more excipients lowers the turbidity and the viscosity of anantisense oligonucleotide composition and also decreases the osmolarityof the antisense composition.

In certain embodiments, one or more excipients may effective reduce theviscosity, turbidity, or both the viscocity and turbidity of a solutionof one or more modified antisense oligonucleotides. In certainembodiments, one or more excipients may effective reduce the viscosity,turbidity, or both the viscocity and turbidity of a solution of one ormore modified antisense oligonucleotides but this excipient may, forexample, increase the osmolality of the solution to an undesirableamount. In certain embodiments,

In certain embodiments, salts such as NaCl, KCl, LiCl, MgCl₂, or CaCl₂may used to modulate the osmolality of an oligonucleotide in solution,but may also induce the turbidity of certain oligonucleotides. Incertain such embodiments, it may therefore be desireable to use anexcipient selected from among L-Tryptophan, Niacinamide,L-Phenylalanine, and L-Histidine to modulate the osmolality of anoligonucleotide solution and to also modulate the viscosity and/orturbidity of the oligonucleotide solution. In certain such embodiments,it may therefore be desireable to use an excipient selected from amongL-Tryptophan, Niacinamide, L-Phenylalanine, and L-Histidine to increasethe osmolality of an oligonucleotide solution and to also decrease theviscosity of the oligonucleotide solution. In certain such embodiments,it may therefore be desireable to use an excipient selected from amongL-Tryptophan, Niacinamide, L-Phenylalanine, and L-Histidine to increasethe osmolality of an oligonucleotide solution and to also decrease theturbidity of the oligonucleotide solution. In certain such embodiments,it may therefore be desireable to use an excipient selected from amongL-Tryptophan, Niacinamide, L-Phenylalanine, and L-Histidine to increasethe osmolality of an oligonucleotide solution and to also decrease theviscosity and decrease the turbidity of the oligonucleotide solution.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligomeric compound having the nucleobase sequence “ATCGATCG”encompasses any oligomeric compounds having such nucleobase sequence,whether modified or unmodified, including, but not limited to, suchcompounds comprising RNA bases, such as those having sequence “AUCGAUCG”and those having some DNA bases and some RNA bases such as “AUCGATCG”and oligomeric compounds having other modified or naturally occurringbases, such as “AT^(me)CGAUCG,” wherein ^(me)C indicates a cytosine basecomprising a methyl group at the 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the presentinvention and are not limiting. Moreover, where specific embodiments areprovided, the inventors have contemplated generic application of thosespecific embodiments. For example, disclosure of an oligonucleotidehaving a particular motif provides reasonable support for additionaloligonucleotides having the same or similar motif. And, for example,where a particular high-affinity modification appears at a particularposition, other high-affinity modifications at the same position areconsidered suitable, unless otherwise indicated.

Example 1

General Method for Evaluating Turbidity, and Viscosity

The general method for measuring turbidity is as follows. Turbidityqualitative assessment is performed by visually inspecting control andsample vials that have frozen at −20° C. and subsequently thawed at 5°C. Their appearance is noted before the samples equilibrate to roomtemperature. These results are often documented with photographs.Turbidity quantitative measurement is performed on a Hach 2100 ANLaboratory Turbidimeter and is used to test turbidity of 3.2-3.4 mL ofsolution filled in a 13-mm glass tube. Typically the solution hasreceived freeze-thaw treatment similar to the one described above toinduce turbidity formation prior to measurement. The instrument isstandardized with Formazin reference suspensions over a range ofNephelometric Turbidity Units (NTUs) that brackets the turbidity of thesamples being tested.

Viscosity qualitative assessment is performed by visually inspecting theflow of a sample solution in a container and comparing it to a control.In this case, sample viscosities were noted as increased or decreasedonly when an obvious difference was noted (e.g., if at 5° C. the controlsample appeared to mimic honey in viscosity, the viscosity of excipientsamples appearing as free-flowing liquids are noted as “decreased”,while excipient samples appearing as solid gel are noted as“increased”). Quantitative viscosity measurement comprises of two forms,dynamic and kinematic. The form relating to antisense oligonucleotide(ASO) drug product characterization is dynamic viscosity. Twoinstruments that are currently used in the Pharmaceutical Development(PD) laboratory to measure dynamic viscosity are RheoSense m-VROCviscometer and Malvern Instruments Bohlin CVO 100 rheometer. Furtherinformation about the instruments can be obtained from themanufacturer's websites (rheosense.com and malvern.com/CVO).

RheoSense m-VROC System

The RheoSense m-VROC utilizes a microelectromechanical systems (MEMS)chip which consists of three silicone pressure sensor arrays embeddedlengthwise along the center of a rectangular channel. The liquid sampleis loaded into a syringe pump, which dispenses the sample into themicrofluidic chip at a specified flow rate. The pressure drop overdistance of a flowing test liquid is measured, and it is expected to belinear for Newtonian fluids if a fully developed flow is ensured withinthe channel. The shear stress and viscosity are calculated according tofluid dynamic principles.

There are four classifications of chips available for the RheoSensem-VROC, labeled A, B, C, and D. They vary consecutively in size, withthe A chip having the smallest inner channel dimensions that enablesprecise measurements of low viscosity samples, while the D chip is builtfor high viscosity samples. Within each chip category there are alsothree channel depths: 20, 50, and 100 μm, indicated as the “02”, “05”,and “10” series. The maximum recommended sample viscosities for use witheach chip are shown in Table 2.

TABLE 2 Recommended upper viscosity limits for each Rheosense m-VROCchip Chip Maximum Recommended Viscosity (cP) A 100 B 400 C 3,000 D10,000Malvern Instruments Bohlin CVO 100 System

The Bohlin CVO 100 operates via a bob-and-cup mechanism, whereby thetorque required to accelerate a bob in a cup filled with sample fluid toa specified angular speed is measured and converted mathematically toviscosity. A single interface is used for all sample types evaluated in,with the bob and cup being removable for sample loading and cleaning.The instrument specifications are shown in Table 3.

TABLE 3 Instrument Specifications for Bohlin CVO 100 System Method Shearramp method with a spinning bob Description rheometer and no inertialcorrections Cell 1 mL Mooney-Ewart Gap 75 microns TemperatureIsothermal, typically at 25° C., 15° C., or 5° C. Standard and 1.2 mLSample Volume Standards 10, 50, and 100 cP polydimethylsiloxaneviscosity reference fluids Model Fit Newtonian

Example 1a

General Method for Evaluating Osmolality

Osmolality is measured using Wescor VAPRO 5600 Vapor Pressure Osmometer.A small paper disc is loaded onto the sampling area, and 10 uL of testsolution is dispensed into the disc. Calibration is performed using 100,290, and 1000 mOsm/kg standards. Further information about thisinstrument can be obtained from the manufacturer's website(wescor.com/biomedical/osmometer/vapro5600.html).

Wescor VAPRO 5600 Vapor Pressure Osmometer

VAPRO 5600 osmometer measures a test solution's dew point temperaturedepression, which is related to its vapor pressure. Vapor pressure is acolligative property of a solution which linearly correlates to theconcentration of particles dissolved in the solvent (i.e. osmolality).The particle's size, density, configuration, or electrical charge has nobearing on a colligative property. Increasing osmolality decreases thesolution vapor pressure.

In the VAPRO system, an internal thermocouple hygrometer joins with thesample holder when the sample has been loaded to form a small chamberenclosing the sample disc. After the chamber temperature hasequilibrated, the thermocouple is cooled, thus inducing dew formation onthe thermocouple surface. The thermocouple is then heated until itreaches the dew point. The difference between ambient temperature andthe dew point temperature is the dew point temperature depression. Theinstrument processes this result and provides a reading in mmol/kg,equaling mOsm/kg. (Note: mOsm/kg refers to the concentration ofdissociated molecules in solution. Therefore, 1 mmol/L NaCl yields 2mmol/L of dissociated ions; i.e. Na⁺ and Cl⁻, which equals 2 mOsm/L.Applying the density conversion of 1 kg/L yields 2 mOsm/kg). Theinstrument specifications are shown in Table 3a.

TABLE 3a Instrument Specifications for Wescor VAPRO 5600 Sample Volume10 μL Measurement Range 20-300 mmol/kg Measurement Time 90 secondsResolution 1 mmol/kg Calibration Opti-mole ™ osmolality standards

Example 2

General Method Used to Screen Excipients for the Mitigation of Turbidityand Viscosity

Turbidity Screening

The screening of the excipients for mitigating turbidity is performed byadding 0.1 to 5% (w/v) of the excipient at pH 7-8 to a solution of ASOin water at a concentration of 200 mg/mL to 250 mg/mL. After thesolution is prepared, a 1 mL sample is aliquoted and filled into a 2-mLclean glass vial, stoppered, sealed, frozen at −20° C., and thawed at 5°C. The sample is then evaluated for turbidity with the turbidimeter ormore commonly by visual inspection. The results from visual inspectionis converted to a score from 0 to 3 with 0 being visually clear (i.e.,<20 NTU); 1 being less turbid than a control but not clear; 2approximately the same turbidity as a control; and 3 being more turbidthan a control. A solution of ASO in water is used as the control.

The excipients investigated for mitigation of turbidity areindependently selected from but are not limited to adenine, benzylalcohol, m-cresol, cytidine, cytidine monophosphate, cytosine, dextran,guanine monophosphate, D-mannitol, methylparaben, nicotinamide, phenol,2-phenoxyethanol, L-phenylalanine, pyridoxine, sodium chloride, thymine,L-tryptophan, L-tyrosine, ascorbic acid, benzamide, o-benzenediol,benzenehexol, caffeine, L-histidine, hydroxypyridine, indole, 2H-pyran,pyrazinamide, pyridine, pyrimidine, 2-pyrone, riboflavin, thiamine,thiamine hydrochloride, 1,2-dihydroxybenzene (catechol), ethylenediamine tetraacetic acid (EDTA), tryptamine, calcium folinate, sodiumfolinate (vitamin B9), D-mannitol and phenol mixture, D-mannitol andpyridine mixture, D-mannitol and niacinamide (nicotinamide) mixture,L-phenylalanine and pyridoxine mixture, pyridoxine and benzyl alcoholmixture, L-phenylalanine and L-histidine mixture; any mixturecombination of L-phenylalanine, L-tryptophan, L-histidine, andniacinamide; and dinucleotides or trinucleotides or other shortmeroligonucleotide probes; and nucleic acid stains such as acridine orange,ethidium bromide, propidium iodide, and cyanine dyes such as PicoGreen®.

Viscosity Screening

A concentrated stock solution of ASO in water is diluted using solidexcipient or a concentrated stock solution of excipient. A typicaldilution is from a concentration of 250 to 200 mg/mL of ASO at pH 8.Viscosity would either be visually noted, or measured using theRheoSense m-VROC viscometer or Malvern Instruments Bohlin CVO 100rheometer as described in Example 1. The measured results for viscosityare then obtained and normalized to the results when the diluent is onlywater with no excipient present.

The excipients investigated for mitigation of viscosity areindependently selected from but are not limited to benzyl alcohol,m-cresol, cytidine, cytosine, dextran, ethanol,(2-Hydroxypropyl)-β-cyclodextrin, D-mannitol, niacin, nicotinamide,polyethylene glycol 600 (PEG₆₀₀), polyethylene glycol 4600 (PEG₄₆₀₀),propylene glycol, pyridoxine, sodium chloride, sucrose, thymidine,L-tryptophan, uridine, adenine, thymine, Tween 80, cytidinemonophosphate, guanine monophosphate, methylparaben, phenol,2-phenoxyethanol, L-phenylalanine, L-tyrosine, ascorbic acid, benzamide,o-benzenediol, benzenehexol, caffeine, L-histidine, hydroxypyridine,indole, 2H-pyran, pyrazinamide, pyridine, pyrimidine, 2-pyrone,riboflavin, thiamine, tryptamine, thiamine hydrochloride,1,2-dihydroxybenzene (catechol), ethylene diamine tetraacetic acid(EDTA), calcium folinate, sodium folinate (vitamin B9), D-mannitol andphenol mixture, D-mannitol and pyridine mixture, D-mannitol and phenolmixture, D-mannitol and pyridine mixture, D-mannitol and niacinamide(nicotinamide) mixture, L-phenylalanine and pyridoxine mixture,pyridoxine and benzyl alcohol mixture, L-phenylalanine and L-histidinemixture; any mixture combination of L-phenylalanine, L-tryptophan,L-histidine, and niacinamide; and dinucleotides or trinucleotides orother shortmer oligonucleotide probes; and nucleic acid stains such asacridine orange, ethidium bromide, propidium iodide, and cyanine dyessuch as PicoGreen®.

Note: Not all the aforementioned excipients are viable for formulation.Some are used in the screening simply for mechanistic studies.

Example 3

Turbidity and Viscosity Evaluation for ISIS NO. 426115

Antisense oligonucleotide Isis No. 426115 was selected for turbidity andviscosity evaluation. The ASO and its motif are described in Table 4.The internucleoside linkages throughout each modified oligonucleotideare phosphorothioate linkages (P═S). Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicate 2′-O-methoxyethyl (MOE) modified nucleosides. An “N” indicatesa U, T, C, ^(me)C, G or A nucleoside.

TABLE 4 Antisense Oligonucleotide Isis No. 426115 Selected for Turbidityand Viscosity Evaluation Isis No. Composition (5′ to 3′) Motif SEQ IDNo. 426115 N_(e)N_(e)N_(e)N_(e)N_(e)NNNNNNNNNNN_(e)N_(e)N_(e)N_(e)N_(e)5-10-5 14Turbidity Evaluation for ISIS NO. 426115

Several excipients in Example 2 were selected and screened for theireffect in mitigating the turbidity of ISIS NO. 426115. The turbidityexperiment was performed in the same manner as described in Example 2.To an aqueous solution of ISIS NO. 426115 at a concentration of 220mg/mL, the excipient was added at the percentage (%, w/v) as indicatedin Table 5. The solution was frozen at −20° C., thawed to 5° C. andsubjected to turbidity evaluation. Turbidity was analyzed and comparedto a control by visual inspection using a scoring format of 0 to 3 with0 being visually clear; 1 being less turbid than a control but notclear; 2 approximately the same turbidity as a control; and 3 being moreturbid than a control. A solution of ISIS NO. 426115 at a concentrationof 220 mg/mL at pH 7-8 in only water was used as the control. Theresults are presented in Table 5.

As illustrated, several excipients when used individually or incombination with other excipients at various concentrations demonstrateddesirable reduction in turbidity with a score of 1 or lower as comparedto the control.

TABLE 5 Effect of various excipients on turbidity for ISIS NO. 426115 at220 mg/mL at 5° C. Excipient concentration Turbidity Excipient (%, w/v)(visual inspection) Adenine 0.5 0 Ascorbic acid 0.5 2 Benzyl alcohol 0.52 1.5 0 Caffeine 0.5 0 1.5 0 m-Cresol 0.3 3 Cytidine 0.01 2 0.2 0.5 5.00 Cytidine Monophosphate 0.5 2 Cytosine 0.005 2 0.2 2 0.5 0 1.0 Dextran0.5 2 1,2-Dihydroxybenzene 0.5 3 (catechol) Ethylene diamine 0.2 2tetraacetic acid (EDTA) 1.0 3 Guanine Monophosphate 0.5 2 L-Histidine1.5 0 2.0 0 Hydroxypyridine or 2 0.5 1 Pyridone 1.0 0 D-Mannitol (0.5) +1.0 (total) 2 Phenol (0.5) mixture D-Mannitol (0.5) + 1.0 (total) 1Pyridine (0.5) mixture D-Mannitol (0.5) + 1.0 (total) 1 Niacinamide(0.5) mixture D-Mannitol 2.0 2 5.0 10 15 Methylparaben 0.2 3 0.5 2 1.5*0 *excipient saturated and precipitated Nicotinamide or 0.5 2Niacinamide 1.0 1 1.5 0 2.0 2.5 3.0 5.0 Phenol 0.5 3 2-Phenoxyethanol0.5 3 L-Phenylalanine 0.5 1 1.5 0 Pyrazinamide 0.5 1 1.0 0 Pyridine 0.52 Pyridoxine 1.0 2 1.5 Sodium Chloride 0.5 2 Thiamine HCl 1 0 Thymine0.5 0 L-Tryptophan 0.1 2 0.2 0.3 0.4 1 0.5 0 1.5 0 L-Tyrosine 0.04 2Calcium Folinate 0.5 2 L-Histidine 0.5 2 1.5 1 2 0 Indole 0.1 2 2H-Pyran0.5 2 Pyrimidine 0.5 2 2-Pyrone 0.5 1 Riboflavin (Vitamin B2) 0.025 2D-Mannitol (0.5) +   1 (total) 1 Nicotinamide/Niacinamide (0.5) mixtureL-phenylalanine (1) + 1.5 (total) 0 Pyridoxine (0.5) Pyridoxine (0.5) +  1 (total) 0 Benzyl alcohol (0.5)Viscosity Evaluation for ISIS NO. 426115

Several excipients in Example 2 were selected and screened for theireffect in mitigating the viscosity of ISIS NO. 426115. The viscosityexperiment was performed in the same manner as described in Example 2. Aconcentrated stock solution of ASO in water was diluted using solidexcipient or a concentrated stock solution of excipient. The dilutionwas at a concentration of 220 mg/mL of ISIS NO. 426115 at pH 7-8.Viscosity at 25° C. and 5° C. was measured using the RheoSense m-VROCviscometer as described in Example 1. The results for viscosity wereobtained and normalized to the results of the control when the diluentwas only water. Normalized viscosity was calculated by dividing theviscosity of the excipient present vs the viscosity of the control. Theresults are presented in Table 6.

As illustrated, at 25° C. or 5° C., several excipients when used aloneor in combination with other excepients at various concentrationsdemonstrated desirable reduction in the viscosity with a normalizedviscosity below 1.00.

TABLE 6 Effect of various excipients on viscosity of ISIS NO. 426115 at220 mg/mL at 25° C. and 5 ° C. Excipient Normalized Conc. Viscosity (cP)Viscosity* Excipient (%, w/v) 25° C. 5° C. 25° C. 5° C. None (Control) 079 28,500 1.00 1.00 L-Tryptophan 0.5 73 12,000 0.9 0.4 1.5 64 7,758 0.80.3 Nicotinamide 1.5 50 3,000 0.6 0.1 (Niacinamide) Phenylalanine 1.5 411,604 0.5 0.1 Thiamine HCl 1 95 79,000 1.2 2.8 Pyridoxine HCl (0.5%) + 1106 19,000 1.3 0.7 benzyl alcohol (0.5%) Pyridoxine HCl (0.5%) + 1.5 462,539 0.6 0.1 phenylalanine (1%) Caffeine 1.5 148 35,000 1.9 1.2Histidine 0.5 73 NT 0.9 NT 1.5 63 NT 0.8 NT 2 47 4,759 0.6 0.2Pyrazinamide 1 59 NT 0.7 NT Methylparaben 1.5 120 NT 1.5 NT *Normalizedto ISIS NO. 426115 at concentration of 220 mg/mL in water NT = NotTested

As reported in scientific or patent literature, some additives canmitigate only turbidity or only viscosity (see US 20110098343). Unlikethe literature, results from Tables 4 and 5 demonstrated that bothturbidity and viscosity can be comitigated by a single excipient such asL-tryptophan, benzyl alcohol, L-histidine, L-phenylalanine, andnicotinamide. In certain embodiments, additional excipients that havepotential to mitigate both turbidity and viscosity based on our currentstudy include but are not limited to thymine, adenine, riboflavin,thiamine, and tryptamine since, in certain embodiments, severaleffective excipients have aromatic character (with heterocylicityenhancing their turbidity mitigation property) and an ability to act asa hydrogen bond donor and/or a hydrogen bond acceptor.

Osmolality Measurement

The osmolality of some samples from the screenings above was measuredand compared to the control using the procedure illustrated in Example1a. The results are presented in Table 6a, below.

The molar increase in solution osmolality compared to the control wasexpected to equal the molar amount of excipient added, since no ionicdissociation was expected. For example, adding 73.4 mmol/kg ofL-Tryptophan (which equals to 73.4 mOsm/kg assuming the density is 1kg/L) to a control solution of 431 mOsm/kg, the expected increase inosmolality of the solution would be 73.4 mOsm/kg (which equals to 504.4mOsm/kg total solution osmolality). As illustrated in Table 6a, bothL-tryptophan and niacinamide showed an increase in osmolality less thanexpected at 26 mOsm/kg, and 66 mOsm/kg rather than at 73.4 mOsm/kg and122.8 mOsm/kg, respectively compared to the control. Phenylalanineshowed an increase in osmolality as expected, while histidine showed anincrease in osmolality more than expected at 119 mOsm/kg.

TABLE 6a Effect of various excipients on osmolality for ISIS 426115 at220 mg/mL Expected Measured Excipient Excipient Expected MeasuredOsmolality Osmolality Conc. Conc. Osmolality Osmolality IncreaseIncrease Excipient (%, w/v) (mmol/kg)* (mOsm/kg) (mOsm/kg) (mOsm/kg)(mOsm/kg) Control 0 0.0 431 431 0 0 L-Tryptophan 1.5 73.4 504.4 457 73.426 Niacinamide 1.5 122.8 553.8 497 122.8 66 L-Phenylalanine 1.5 90.8521.8 522 90.8 91 L-Histidine 1.5 96.7 527.7 550 96.7 119 *assumingdensity = 1 kg/L

Example 4

Evaluation of Properties of Effective Turbidity and ViscosityComitigator Excipients for ISIS NO. 426115

The properties of excipients which can effectively comitigate turbidityand viscosity of ISIS NO. 426115 were investigated. Several excipientsin Example 2 were selected and screened using the same method asdescribed previously. The properties that were examined included, butnot limited to aromaticity, homocyclic vs. heterocyclic aromatic rings,and the number of hydrogen bond donor and/or acceptor.

The ASO samples were prepared in the same manner as described in Example3. To an aqueous solution of ISIS NO. 426115 at a concentration of 220mg/mL, the excipient was added at the percentage (%, w/v) as indicatedin Table 7. The solution was frozen at −20° C., thawed to 5° C. andsubjected to turbidity and viscosity evaluation. Turbidity and viscositywere analyzed and compared to a control by visual inspection. Forturbidity evaluation, a scoring format of 0 to 3 was employed with 0being visually clear; 1 being less turbid than a control but not clear;2 approximately the same turbidity as a control; and 3 being more turbidthan a control. A solution of ISIS NO. 426115 at a concentration of 220mg/mL at pH 8 in only water was used as the control. The results arepresented in Table 7.

As illustrated, ascorbic acid reduced turbidity but not viscositycompared to the control, while L-phenylalanine was able to reduce bothturbidity and viscosity. Similarly, pyrazinamide was effective forco-mitigation at 0.5% (w/v). Benzamide was effective for mitigatingturbidity but not for viscosity at 2% (w/v), while nicotinamide was ableto co-mitigate turbidity and viscosity at the same concentration. Phenolwas unable to mitigate turbidity and viscosity, while hydroxypyridinewas able to co-mitigate. Thus, in certain embodiments, heterocyclicitywith increasing number of non-carbon substituents such as nitrogen andoxygen, seem to improve turbidity reduction.

Both phenol and catechol were ineffective in reducing turbidity at 0.5%(w/v). Phenol appeared to increase viscosity while catechol had noeffect. Pyridine was ineffective for both turbidity and viscosityreduction, while hydroxypyridine was effective for co-mitigation. Theseresults suggest that excipients with more hydrogen bond donors and/oracceptors can be effective at turbidity and viscosity co-mitigation.

In certain embodiments, some excipients effective at decreasingturbidity and/or viscosity have heterocyclic or homocyclic aromaticcharacter. In certain embodiments, some excipients effective atdecreasing turbidity and/or viscosity are heterocyclic and nonaromatic,for example, ascorbic acid as shown in Table 7.

The results from Table 7 suggest that in certain embodiments, excipientsthat have heterocyclic aromatic or nonaromatic character and containhydrogen-bond donor(s) and/or hydrogen-bond acceptor(s) mitigate bothturbidity and viscosity.

In certain embodiments, the properties given in this example areconsistent with single excipients that are effective at mitigating bothturbidity and viscosity, for example, compounds that resemblenucleobases, and heterocyclic compounds that possess both hydrogen bonddonors and acceptors. In certain embodiments, aromatic ring characterappears to provide a benefit based on the planar nature thatfacilitates: (i) positioning hydrogen bonds at a low energy state and(ii) interference of base stacking.

TABLE 7 Effect of excipients in mitigating turbidity and viscosity ofISIS NO. 426115 Viscosity Compared to Turbidity Control Concentration(visual (visual Chemical Structure Excipient (%, w/v) inspection)inspection)

Phenol 0.5 3 Increased

Catechol (1,2- Dihydroxybenzene) 0.5 3 No notable difference

Hydroxypyridine 0.5 1 Decreased

Benzamide 2   3 Decreased

Nicotinamide 0.5   2   2   0 No notable difference Decreased

Pyrazinamide 0.5 1 Decreased

Pyridine 0.5 2 No notable difference

Ascorbic acid 0.5 0 No notable difference

Phenylalanine 0.5 1 Slightly decreased

Example 5

Effect of Combining Singly Ineffective Excipients for Co-Mitigation ofISIS NO. 426115

The effect of combining excipients which are ineffective by themselvesat mitigating both turbidity and viscosity of ISIS NO. 426115 wasinvestigated. Several excipients in Example 2 were selected and screenedusing the same method as described previously. The ASO samples wereprepared in the same manner as described in Example 3. To an aqueoussolution of ISIS NO. 426115 at a concentration of 220 mg/mL at pH 8 theexcipient mixture was added at the percentage (%, w/v) indicated inTable 8. The solution was frozen at −20° C., thawed to 5° C. andsubjected to turbidity and viscosity evaluation. Turbidity and viscositywere analyzed and compared to a control by visual inspection. Forturbidity evaluation, a scoring format of 0 to 3 was employed with 0being visually clear; 1 being less turbid than a control but not clear;2 approximately the same turbidity as a control; and 3 being more turbidthan a control. A solution of ISIS NO. 426115 at a concentration of 220mg/mL at pH 8 in only water was used as the control. The results arepresented in Table 8.

In certain embodiments, the chemical properties listed previously namely(i) aromatic homocyclicity or heterocyclicity and (ii) the ability toact as a hydrogen bond donor and/or acceptor do not have to beconsolidated into one excipient or compound. In certain embodiments, itwas discovered from our finding that a mixture of two excipients, whichsatisfies the two properties as a whole, can also be effective inmitigating both turbidity and viscosity. In certain embodiments, thiswas demonstrated by the mixture of 0.5% (w/v) mannitol and 0.5% (w/v)pyridine. Mannitol is a saturated linear carbon chain with hydroxylgroups, therefore possessing both hydrogen bond donors and acceptors;whereas pyridine is a heterocyclic aromatic compound lacking a hydrogenbond donor but has a hydrogen bond acceptor from its nitrogen atom. Thismixture visually reduced both turbidity and viscosity compared to thecontrol. In contrast, mannitol and pyridine by itself are ineffective atmitigating either turbidity or viscosity. In support of our observationfor chemical properties of the excipients, it was found that mannitoland phenol mixture lacking a heterocyclic ring showed no significantmitigation in turbidity (Table 5).

The results from Table 8 suggest that in certain embodiments, othermixtures may potentially have such synergistic effects for bothturbidity and viscosity mitigation as demonstrated by D-mannitol andpyridine mixture. Such mixtures include, but are not limited to, dextranand pyrimidine mixture or ascorbic acid and phenanthroline mixture.

TABLE 8 Effect of excipient mixtures in mitigating turbidity andviscosity of ISIS NO. 426115 Viscosity Compared to Turbidity ControlConcentration (visual (visual Chemical Structure Excipient (%, w/v)inspection) inspection)

Pyridine 0.5 2 No notable difference

D-Mannitol 2   5   10   15   2 No notable difference See boxes aboveD-Mannitol + Pyridine 0.5 + 0.5 1 Decreased

Example 6

Effect of Temperature and Time Dependency on ISIS NO. 426115 Turbidity

The effect of temperature and time dependency on the turbidity profileof freeze-thawed ISIS NO. 426115 at 220 mg/mL as it warmed up from 5° C.to room temperature was evaluated. The experiment was performed in thefollowing manner.

Materials

The materials used to carry out the experiment included a solution ofISIS NO. 426115 in water at a concentration of 220 mg/mL and 150 mg/mL;a Hach 2100AN Nephelometer; a StablCal Calibration Set for 2100ANNephelometer, a StablCal Formazin Turbdity Standard at 1000 NTU; a VWRPrecision 0.01° thermometer and Gerresheimer 13×100 mm glass culturetubes.

Method

A 4 mL sample of ISIS NO. 426115 at concentration of 220 mg/mL or 150mg/mL was pipetted into a sample tube, capped and parafilmed at the capjoint. Both samples were allowed to freeze in a −20° C. storage and thenmoved to a 2-8° C. storage to thaw.

Turbidity Measurement

The turbidimeter was calibrated using Formazin turbidity calibrationset. The 220 mg/mL sample from 2-8° C. storage was removed and allowedto stand at room temperature. The temperature of the sample wasmonitored until it reached approximately 13° C. or until condensationdoes not recur upon wiping of the tube surface and the temperature wasrecorded. The tube was then inserted in the nephelometer using a tubeadaptor and turbidity measurement was taken every 2 minutes untilturbidity value does not change by more than 0.1 NTU.

Temperature Profile

The 150 mg/mL sample from 2-8° C. storage was removed and allowed tostand at room temperature. The sample tube was inserted into thenephelometer (which does not need to be turned on). Condensation was notaccounted for since it did not significantly affect the temperatureprofile. A temperature probe was inserted into the sample tube and thetemperature was recorded over time.

Note: The temperature profiles of 150 mg/mL and 220 mg/mL samples ofISIS NO. 426115 has been shown to be identical in previous studies.

Sample Tube Turbidity Correction Factor (CF)

Since the 13-mm sample tubes used differ from the nephelometer'srecommended set, a different path length of light applies for taking thesample turbidity. As such a CF was required to convert the readings intoaccurate NTU (Nephelometric Turbidity Units) values.

Formazin turbidity standards were volumetrically prepared by serialdilution of 1000 NTU standard using sterile water for injection. Thestandard values are 0, 100, 250, 500, 750 or 1000 NTU. The standardsolutions were filled into 13-mm sample tubes and turbidity wasmeasured. The standard values were plotted over measured turbidityvalues and a linear trend over the data points were fitted to obtain theslope. The slope is the CF for 13-mm sample tubes. The CF was used toconvert measured values from 220 mg/mL ISIS NO. 426115 sample readingsinto actual NTU values.

Data Analysis and Results

The temperature and turbidity measurements over time were tabulated. Thetemperature profile was plotted by designating time zero to be at thesame starting temperature as the turbidity sample. Overlaid theturbidity and temperature profiles over time and matched up theturbidity value with a temperature value along the time profile togenerate an approximate turbidity vs temperature profile (FIG. 1 ).

The results showed that the CF for the tubes is the slope of the plotwhich is 1.8148 (FIG. 1 ). A decrease in turbidity appeared to lagbehind the increase in temperature as shown in FIGS. 2 and 3 . Turbidityremained at approximately 1800 NTU for the first 10 minutes when thetemperature had increased by about 5 degrees. The solution temperaturereached room temperature after approximately 30 minutes, howevervisually the turbidity did not completely dissipate until approximately50 minutes, when it reached 20 NTU. FIG. 4 shows that 20 NTU is theapproximate lower limit of visible turbidity in a solution.

In certain embodiments, this observation is indicative of a certainamount of thermal energy being required to break up the turbid species,which has been hypothesized to be self associated oligonucleotides. Inaddition, in certain embodiments, as shown in FIG. 3 , there is anapparent melting temperature existing at approximately 19° C. Attemperatures below that, the turbid species persist, and at temperaturesabove that they dissociate.

Example 7

Viscosity Evaluation for ISIS NO. 104838

Antisense oligonucleotide Isis No. 104838 was selected for viscosityevaluation. The ASO and its motif are described in Table 9. Theinternucleoside linkages throughout each modified oligonucleotide arephosphorothioate linkages (P═S). Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”indicate 2′-O-methoxyethyl (MOE) modified nucleosides. An “N” indicatesa U, T, C, ^(me)C, G or A nucleoside.

Several excipients in Example 2 were selected and screened for theireffect in mitigating the viscosity of ISIS NO. 104838. The viscosityexperiment was performed in the same manner as described in Example 2. Aconcentrated stock solution of ASO in water was diluted using solidexcipient or a concentrated stock solution of excipient. The dilutionwas at a concentration of 200 mg/mL of ISIS NO. 104838 at pH 8.Viscosity at 25° C. was measured using the Malvern Instruments BohlinCVO 100 rheometer as described in Example 1. The results for viscositywere obtained and normalized to the results of the control when thediluent was only water. The range of the control viscosity (105.1-113.6cP) provided in Table 10 was generated from various independent studiesand therefore, normalized viscosity was calculated based on the controlvalue obtained from the same study. The results are shown below.

As illustrated, several excipients at various concentrationsdemonstrated a desirable reduction in viscosity with a normalizedviscosity of below 1.00 as compared to the control.

TABLE 9 Antisense Oligonucleotide Isis No. 104838 Selected for ViscosityEvaluation Isis No. Composition (5′ to 3′) Motif SEQ ID No. 104838N_(e)N_(e)N_(e)N_(e)N_(e)NNNNNNNNNNN_(e)N_(e)N_(e)N_(e)N_(e) 5-10-5 26

TABLE 10 Effect of various excipients on viscosity of ISIS NO. 104838 at200 mg/mL at 25° C. Excipient Conc. Viscosity Normalized Excipient (%,w/v) (cP) Viscosity* None (control) 0 105.1-113.6 1.00 PEG₄₆₀₀ 5 194.81.85 (2-Hydroxypropyl)- 5 178.2 1.60 β-cyclodextrin PEG₆₀₀ 5 147.2 1.40Dextran 1500 5 130.9 1.18 D-Mannitol 5 115.5 1.10 Sucrose 5 122.1 1.10Niacin (Vitamin B₃) 5 123.8 1.09 sodium salt Tween 80 0.5 115.1 1.04Thymidine 5 94.75 0.86 Uridine 5 89.05 0.81 Cytosine 1 81.89 0.74L-Tryptophan 1.1 82.69 0.73 Benzyl alcohol 0.9 82.09 0.72 3.8 44.08 0.39Cytidine 5 67.64 0.61 m-Cresol 2 62.40 0.55 Nicotinamide/Niacinamide 542.46 0.38 *Normalized to ISIS NO. 104838 concentration of 200 mg/mL inwater

Example 8

Combining Singly Effective Excipients for Enhanced Turbidity andViscosity Co-Mitigation of ISIS NO. 426115

The effect of combining effective excipients was evaluated forco-mitigation of turbidity and viscosity of ISIS NO. 426115 beyond whateach excipient could perform individually. Tryptophan, niacinamide(nicotinamide), L-phenylalanine and L-histidine were selected for afactorial mixture design-of-experiment (DOE) study, where the total ofall excipient mixture combinations equal 60 mM. The ratios are presentedin Table 11, below.

Stock solutions of 220 mg/mL ISIS NO. 426115 containing 60 mM singleexcipients were prepared by adding solid ASO and excipient powders,diluting with water, and then pH-adjusting to target pH 7-8 asnecessary. Subsequently, the stock solutions were combined in relevantratios to total 1 mL solutions, and filled into 2-mL clean glass vialswhich were stoppered and sealed, to produce all 24 samples. Controlsolution of 220 mg/mL ISIS NO. 426115 without excipient was preparedseparately using similar method. These samples were frozen and thawed intwo cycles, by first freezing at −20° C. and thawing at 5° C., and thenthe process was repeated. Turbidity was analyzed after the twofreeze-thaw cycles and compared to the control by visual inspection. Ascoring format of 0 to 2 was employed with 0 being visually clear; 0.5being very slightly turbid, 1 being slightly turbid; and 2 being asturbid as the control. Viscosity was measured at 25° C. using theRheosense m-VROC system as described in Example 1. The results forviscosity were obtained and normalized to the results of the controlwhen the diluent was only water. The results presented in Table 11 werethen entered into a DOE software (Design-Expert®8) to determinestatistical significance of the data and render a response surfaceshowing the most ideal mixture for viscosity reduction.

As illustrated, all combinations of excipients at the ratios testedmitigated turbidity with the score of 1 or lower and also mitigatedviscosity to less than 65% of the control. Design-Expert ANOVA analysisdetermined that the results possess adequate signal-to-noise ratio, andcould be used to generate statistically significant models.Additionally, a viscosity response surface generated by the softwareshowed that 50/50 combination of L-phenylalanine and L-histidine provedmost effective at lowering viscosity.

TABLE 11 Effect of excipient mixtures in mitigating turbidity andviscosity of ISIS NO. 426115 at 25° C. L- Nicotinamide L- L- TurbiditySample tryptophan (Niacinamide) phenylalanine histidine (visualViscosity Normalized Type (mM) (mM) (mM) (mM) inspection) (cP) ViscosityControl 0 0 0 0 2 66.841* 1.0 Test 60 0 0 0 0 38.855 0.58 Samples 0 60 00 0 35.254 0.53 0 0 60 0 0 34.296 0.51 0 0 0 60 1 34.863 0.52 30 30 0 00 36 0.54 30 0 30 0 0 33.306 0.50 30 0 0 30 0 38.378 0.57 0 30 30 0 035.216 0.53 0 30 0 30 0.5 35.61 0.53 0 0 30 30 0.5 29.147 0.44 20 20 200 0 35.863 0.54 20 20 0 20 0 36.114 0.54 20 0 20 20 0 32.279 0.48 0 2020 20 0.5 32.829 0.49 15 15 15 15 0 34.739 0.52 38 8 8 8 0 37.625 0.56 838 8 8 0 34.778 0.52 8 8 38 8 0 26.026 0.39 8 8 8 38 0.5 35.13 0.53 60 00 0 0 41.662 0.62 0 60 0 0 0 36.625 0.55 0 0 60 0 0 34.082 0.51 0 0 0 601 35.56 0.53 30 30 0 0 0 37.494 0.56 *Measured by taking the averagefrom three independent studies

Example 10

Turbidity Evaluation for ISIS 442245 in the Presence of OsmolalityAdjusters

Another ASO sequence, ISIS 442245 was used for turbidity evaluation.ISIS 442245 and its motif are described in Table 12. The internucleosidelinkages throughout each modified oligonucleotide are phosphorothioatelinkages (P═S). Nucleosides without a subscript areβ-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “g”indicate 3′-F-HNA modified nucleosides. An “N” indicates a U, T, C,^(me)C, G or A nucleoside.

Osmolality adjusters for ISIS 442245 formulated at low ASO concentrationcan comprise salts such as NaCl, or sugars such as mannitol, or othersubstances. The effect of adding 0.6% (w/v) NaCl to 55 mg/mL ISIS 442245was compared to adding 0.4% (w/v) mannitol. A buffered aqueous solutionof ISIS 442245 at a concentration of 55 mg/mL was prepared by addingsolid drug substance powder to 5 mM phosphate buffer (pH 7-8). To thisstock solution, either 0.4% (w/v) mannitol or 0.6% NaCl (w/v) was added.A control solution was also prepared without osmolality adjusters.

The solutions were frozen at −20° C., thawed to 5° C. Turbidity wasevaluated by visual inspection and noted as either “clear” or “turbid”.The results are presented in Table 13, below.

As illustrated, only ISIS 442245 with NaCl solution became turbid afterfreeze-thaw. The solution containing mannitol remained clear, whichsuggests that mannitol or other sugars such as glucose, sucrose, orfructose, may likely be suitable for use as osmolality adjustersreplacing NaCl for turbidity mitigation.

TABLE 12 Antisense Oligonucleotide Isis 442245 Selected for TurbidityEvaluation Isis No. Composition (5′ to 3′) Motif SEQ ID No. 442245N_(g)N_(g)NNNNNNNNNNN_(g)N_(g) 2-10-2 27

TABLE 13 Turbidity Evaluation of ISIS 442245 in the Presence or Absenceof Osmolality Adjusters Samples Turbidity (all buffered with 5 mMOsmolality (visual phosphate buffer, pH 7-8) (mOsm/kg) inspection)Control: 55 mg/mL 442245 274 Clear 55 mg/mL 442245 + 0.4% mannitol 505Clear 55 mg/mL 442245 + 0.6% NaCl 488 Turbid

Example 11

Turbidity Evaluation for ISIS 442245 with Excipients

Excipients such as tryptophan, niacinamide (nicotinamide) andphenylalanine were selected and screened for their effect in mitigatingthe turbidity of ISIS 442245 formulated with 5 mM phosphate buffer (pH7-8) and 0.6% NaCl. The turbidity experiment was performed in the samemanner as described in Example 2. To an aqueous solution of ISIS 442245at a concentration of 55 mg/mL the excipients were added at thepercentage (%, w/v) indicated in Table 14. L-Tryptophan andL-Phenylalanine concentrations tested were limited by their solubility.The solutions were frozen at −20° C., thawed to 5° C. and subjected toturbidity evaluation. Turbidity was analyzed and compared to a controlby visual inspection using a scoring format of 0 to 3 with 0 beingvisually clear; 1 being less turbid than a control but not clear; 2approximately the same turbidity as a control; and 3 being more turbidthan a control. A solution of ISIS 442245 at a concentration of 55 mg/mLwith 5 mM phosphate buffer (pH 7-8) and 0.6% NaCl is used as thecontrol. The results are presented in Table 14.

As illustrated, the control appeared to be a turbid gel at 5° C.L-phenylalanine was shown to be effective in co-mitigating turbidity andviscosity at approximately 200 mM when the solution was stored forseveral days in 5° C. Upon freeze-thaw, the saturated L-phenylalanineprecipitated and therefore caused the solution to become more turbidthan the control. L-Tryptophan was also effective at turbiditymitigation at 50 mM after freeze-thaw, and would likely be a successfulco-mitigator if it had been more soluble. Further, niacinamide was shownto co-mitigate turbidity and viscosity successfully at 400 mM whileremaining stable after freeze-thaw. The results demonstrate that theseexcipients can be effective at mitigating turbidity and/or viscosity andcan be used as co-mitigators for oligonucleotide sequences other thanthe ones exemplified herein.

TABLE 14 Effect of various excipients on turbidity and viscosity of ISISNO. 442245 at 55 mg/mL formulated with 5 mM buffer pH 7-8 and 0.6% NaClTurbidity Turbidity Excipient after 3 days at after freeze- Viscosity atViscosity Conc. Excipient 5° C. (visual thaw (visual 5° C. (visual at 5°C. Excipient (% w/v) Conc. (mM) inspection) inspection) inspection) (cP)Control 0 0 2 2 N/A See note** Niacinamide 4.9 400 0 0 Decreased 5L-Tryptophan 1.0 50 2 1 No notable See note** difference L-Phenylalanine3.3 * 200* 0 3 Decreased 31 2.5 150 0 2 Decreased NT *Due to excipientsaturation, some excipients precipitated at this concentration**Viscosity traces were erratic, showing some maximum values above 100cP and minima around 0 cP. This is likely due to inhomogeneous samplegelling. NT = not tested

The invention claimed is:
 1. A method of reducing the turbidity,viscosity, or both viscosity and turbidity of an antisenseoligonucleotide solution, said method comprising adding at least oneexcipient to an antisense oligonucleotide solution having an antisenseoligonucleotide present at a concentration of 100-500 mg/ml wherein theantisense oligonucleotide comprises a modified oligonucleotidecomprising at least one modified nucleoside, and wherein the viscosityand/or turbidity of the antisense oligonucleotide solution containingthe excipient(s) is less than the viscosity and/or turbidity of theantisense oligonucleotide solution that does not contain theexcipient(s).
 2. The method of claim 1, wherein at least one of the atleast one excipient comprises an aromatic ring or a heterocyclic ringcompound.
 3. The method of claim 1, wherein at least one of the at leastone excipient is selected from the group consisting of adenine, benzylalcohol, m-Cresol, cytidine, cytidine monophosphate, cytosine, dextran,guanine monophosphate, Methylparaben, nicotinamide, Phenol,2-Phenoxyethanol, L-phenylalanine, pyridoxine, Sodium Chloride, thymine,tryptophan, L-tryptophan, L-tyrosine, Ascorbic Acid, benzamide,o-Benzenediol, benzenehexol, L-histidine, hydroxypyridine, indole,D-Mannitol+Phenol mixture, D-Mannitol+Pyridine mixture, 2H-pyran,pyrazinamide, pyrimidine, 2-pyrone, riboflavin, thiamine, tryptamine,ethanol, (2-Hydroxypropyl)-β-cyclodextrin, Niacin, Polyethylene Glycol600, Polyethylene Glycol 4600, Propylene Glycol, Sucrose, thymidine,Tween 80, uridine, caffeine, acridine orange, ethidium bromide,propidium iodide, a cyanine dye, thiamine hydrochloride, ethylenediamine tetraacetic acid, 1,2-dihydroxybenzene, 1,2-dihydroxybenzene(catechol), D-Mannitol+niacinamide mixture, calcium folinate,L-phenylalanine+pyridoxine mixture, pyridoxine+benzyl alcohol mixture,pyridoxine HCl+benzyl alcohol mixture, niacin sodium salt, dextran 1500,and pyridoxine HCl+phenylalanine mixture.
 4. The method of claim 1,wherein at least one of the at least one excipient increases theosmolarity of the solution, increases the pH of the solution, decreasesthe pH of the solution, and/or buffers the pH of the solution.
 5. Themethod of claim 1, wherein the antisense oligonucleotide solutioncomprises a conjugated antisense oligonucleotide compound.
 6. The methodof claim 1, wherein the antisense oligonucleotide has a dynamicviscosity of more than 40 cP at 25° C., and/or has a turbidity of morethan 20 NTU, when mixed in water or saline solution and in the absenceof the at least one excipient.
 7. The method of claim 1, wherein theantisense oligonucleotide is present at a concentration of 100-220mg/mL.
 8. The method of claim 1, wherein the antisense oligonucleotideis present at a concentration of 140-220 mg/mL.
 9. The method of claim1, wherein the antisense oligonucleotide is present at a concentrationof 160-220 mg/mL.
 10. The method of claim 1, wherein at least one of theat least one excipient comprises a heterocyclic aromatic ring compound.11. The method of claim 1, wherein at least one of the at least oneexcipient comprises a heterocyclic aromatic ring compound comprising anitrogen atom.
 12. The method of claim 1, wherein the at least onemodified nucleoside comprises at least one modified sugar moiety. 13.The method of claim 12, wherein the at least one modified sugar moietyis a 2′-substituted sugar moiety.
 14. The method of claim 13, whereinthe 2′-substituent of the at least one 2′-substituted sugar moiety iseach independently selected from among: 2′-OMe, 2′-F, and 2′-MOE. 15.The method of claim 12, wherein the at least one modified sugar moietyis a bicyclic sugar moiety.
 16. The method of claim 15, wherein the atleast one bicyclic sugar moiety is LNA or cEt.
 17. The method of claim1, wherein at least one of the at least one excipient is adenine,cytidine, cytidine monophosphate, cytosine, guanine monophosphate,nicotinamide, pyridoxine, thymine, L-histidine, hydroxypyridine,pyrazinamide, pyrimidine, thiamine, thymidine, uridine, caffeine, orthiamine hydrochloride.
 18. The method of claim 1, wherein at least oneof the at least one excipient is benzyl alcohol, m-Cresol,L-phenylalanine, L-tyrosine, benzamide, or benzenehexol.
 19. The methodof claim 1, wherein at least one of the at least one excipient isL-tryptophan, indole, riboflavin, tryptamine, or calcium folinate. 20.The method of claim 1, wherein at least one of the at least oneexcipient is 2H-pyran or 2-pyrone.